Biomarkers for predicting and assessing responsiveness of thyroid and kidney cancer subjects to lenvatinib compounds

ABSTRACT

Biomarkers are provided that are predictive of a subject&#39;s responsiveness to a therapy comprising lenvatinib or a pharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate). The biomarkers, compositions, and methods described herein are useful in selecting appropriate treatment modalities for a subject having cancer (e.g., thyroid cancer, kidney cancer), suspected of having cancer, or at risk of developing cancer.

FIELD OF THE INVENTION

The present invention relates generally to biomarkers and thyroid andkidney cancer.

BACKGROUND OF THE INVENTION

A number of kinase inhibitors have been developed as antitumor agents.For example, a group of compounds having inhibitory activity againstreceptor tyrosine kinases, such as vascular endothelial growth factorreceptor (VEGFR), are known to inhibit angiogenesis and are regarded asa new class of antitumor agents. Lenvatinib mesylate (also known asE7080) is an oral tyrosine kinase inhibitor targeting VEGFR1-3,fibroblast growth factor receptor (FGFR) 1-4, rearranged duringtransfection receptor (RET), KIT, and platelet-derived growth factorreceptor (PDGFR). In phase I clinical studies of lenvatinib mesylate,response to treatment was observed in thyroid, kidney and endometrialcancers, as well as melanoma.

Unfortunately, most anti-tumor treatments are associated withundesirable side effects, such as profound nausea, vomiting, or severefatigue. Also, while anti-tumor treatments have been successful, they donot produce significant clinical responses in all patients who receivethem resulting in undesirable side effects, delays, and costs associatedwith ineffective treatment. Therefore, biomarkers that can be used topredict the response of a subject to an antitumor agent prior toadministration thereof are greatly needed. In addition, it is useful tohave biomarkers that can be used to evaluate whether therapy comprisingan antitumor agent is effective.

SUMMARY

The present application is based, at least in part, on theidentification of biomarkers that are predictive of a thyroid or akidney cancer subject's responsiveness to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate). The presence of a mutation in one or more of genesis a useful predictor of responsiveness to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate). For example, a mutation(s) in one or more of thegenes NRAS, KRAS, VHL, BRAF, ERBB2, PTEN, and MET is indicative that agiven thyroid or kidney cancer subject will respond to a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate). In addition, the ratio of thyroglobulinlevels pre- and post-treatment with a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof can be useful in determiningthe likelihood that a subject having differentiated thyroid cancer willrespond to continued therapy with the lenvatinib compound. Furthermore,the expression level of certain proteins (e.g., those listed in Table 3)either prior to or post-treatment, or the ratio of the expression levelpost/pre-treatment compared to a control, can also be a useful predictorof responsiveness to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).Also, the combination of these two classes biomarkers (mutations andblood biomarkers) or three classes biomarkers (mutations, thyroglobulin,and blood biomarkers) can provide for even stronger predictions of thelikelihood that a subject having thyroid or kidney cancer will respondto a therapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate).

The application also provides methods for evaluating whether to continuetreatment with lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate) for a subject having thyroid or kidneycancer. Low or high levels of certain proteins (e.g., those listed inTable 3) before and/or after treatment with the therapy can be useful inevaluating whether to continue treatment with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).For example, lower ratios of thyroglobulin levels (post/pre-treatmentwith lenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate)) compared to control ratios from samples ofpatients who are known to not respond to such therapy can be useful inassessing/evaluating whether the test subject will benefit fromcontinued therapy comprising lenvatinib or a pharmaceutically acceptablesalt thereof (e.g., lenvatinib mesylate).

Thus, the biomarkers and compositions described herein are useful, forexample, in identifying and/or selecting a patient or a subset ofpatients having thyroid or kidney cancer that could benefit fromtreatment with lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate). In addition, the methods described hereinare useful, for example, in selecting appropriate treatment modalities(e.g., therapy comprising lenvatinib or a pharmaceutically acceptablesalt thereof (e.g., lenvatinib mesylate)) for a subject suffering from,suspected of having, or at risk of developing a thyroid or kidneycancer. Also, the methods allow a health care practitioner to determinewhether to continue with a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) orchange therapies and use a different treatment.

In one aspect, the disclosure provides a method of predicting theresponse of a subject having, suspected of having, or at risk ofdeveloping, a thyroid cancer or a kidney cancer to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof. The methodinvolves providing a biological sample obtained from the subject anddetecting the presence of a mutation in at least one gene selected fromthe group consisting of RAS, VHL, and BRAF in the biological sample. Thepresence of a mutation in the at least one gene is predictive that thesubject will respond to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof. In one embodiment, RAS is KRASor NRAS. In one embodiment, the mutation in at least one gene is amutation listed in Table 1. In another embodiment, the mutation in atleast one gene is a mutation listed in Table 2. In another embodiment,the mutation in RAS is selected from the group consisting of KRAS Q61R,KRAS G12R, NRAS Q61P, and NRAS Q61R. In another embodiment, the methodof this aspect further involves detecting the presence of a mutation inat least one gene selected from the group consisting of ERBB2, PTEN, andMET in the biological sample, wherein the presence of a mutated RAS anda mutation in at least one of ERBB2, PTEN, and MET is even more stronglypredictive that the subject will respond to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof. In oneembodiment, the method further comprises the step of determining theexpression level of at least one gene selected from the group consistingof ANGPT2, VEGFA, FLT4, CCL3, and CCL4.

In a second aspect, the application provides another method ofpredicting the response of a subject having, suspected of having, or atrisk of developing, a differentiated thyroid cancer to a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof.This method can also be used to evaluate/assess the benefit of continuedadministration of a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof. The method involves providing a first bloodsample obtained from the subject before the therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof, providing asecond blood sample obtained from the subject after initiation of thetherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof measuring the concentration of thyroglobulin in the first bloodsample and the second blood sample; and calculating the ratio(second/first) of the concentrations of thyroglobulin. A reduced ratio,as compared to a control, of the concentration of thyroglobulin in theblood samples is predictive that the subject will respond to a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof, andan increased ratio, as compared to a control, of the concentration ofthyroglobulin in the blood samples is predictive that the subject willrespond less effectively to the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof than a subject having a reducedratio, as compared to a control, of the concentration of thyroglobulinin the blood samples. In one embodiment, the second blood sample isobtained from the subject 1 week to 9 months after the initiation of thetherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof. In another embodiment, the second blood sample is obtained fromthe subject 2 weeks to 9 months after the initiation of the therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof. Inanother embodiment, the second blood sample is obtained from the subject4 weeks to 6 months after the initiation of the therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof. In a furtherembodiment, the second blood sample is obtained from the subject 4 daysto 2 weeks after the initiation of the therapy comprising lenvatinib ora pharmaceutically acceptable salt thereof.

In a third aspect, the disclosure provides another method of predictingthe response of a subject having, suspected of having, or at risk ofdeveloping, a thyroid cancer or a kidney cancer to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof. This methodinvolves providing a biological sample obtained from the subject beforethe therapy comprising lenvatinib or a pharmaceutically acceptable saltthereof; and measuring the concentration of at least one proteinselected from the group consisting of ANGPT2, VEGFA, IFNG, KDR (solubleVEGFR2), FLT4 (soluble VEGFR3), IL6, PDGFAB, CSF3 (G-CSF), CCL3(MIP-1α), CCL4 (MIP-1ß), FGF2, and IL13 in the biological sample. Areduced concentration, as compared to a control, of ANGPT2, VEGFA, IFNG,or soluble KDR (soluble VEGFR2), and/or an increased concentration, ascompared to a control, of IL-6, IL-13, PDGFAB, CSF3 (G-CSF), CCL3(MIP-1α), CCL4 (MIP-1ß), FLT4 (soluble VEGFR3), or FGF2 is indicativethat the subject will respond to the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof. In one embodiment, theconcentration of at least two genes is measured. In one embodiment, thetwo genes are selected from the group consisting of VEGFA, ANGPT2, andCSF3; or IL13, CCL3 and CCL4. In another embodiment, the concentrationof at least three genes is measured. In yet another embodiment, theconcentration of at least four genes is measured.

In a fourth aspect, the disclosure provides another method of predictingthe response of a subject having, suspected of having, or at risk ofdeveloping, a thyroid cancer or a kidney cancer to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof. This methodcan also be used to evaluate/assess the benefit of continuedadministration of a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof. The method involves providing a biologicalsample obtained from the subject after initiation of the therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof andmeasuring the concentration of at least one protein selected from thegroup consisting of ANGPT2, IL13, VEGFA, IL6, PGF, IL10, CXCL12, andCCL5 in the biological sample. A reduced concentration, as compared to acontrol, of ANGPT2, IL13, VEGFA, IL6, or PGF, and an increasedconcentration, as compared to a control, of IL10, CXCL12 or CCL5 isindicative that the subject will respond to the therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof. In oneembodiment, the sample is obtained about 15 days after initiation of thetherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof. In one embodiment, the sample is obtained about 29 days or anyfirst day of each treatment cycle (four weeks per cycle) afterinitiation of the therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof.

In a fourth aspect, the disclosure provides another method of predictingthe response of a subject having, suspected of having, or at risk ofdeveloping, a thyroid cancer or a kidney cancer to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof. This methodcan also be used to evaluate/assess the benefit of continuedadministration of a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof. The method involves providing a firstbiological sample obtained from the subject before the therapycomprising lenvatinib or a pharmaceutically acceptable salt thereofproviding a second biological sample obtained from the subject afterinitiation of the therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof measuring the concentration of at least oneprotein selected from the group consisting of CCL5, FLT3LG, IL12(p40),EGF, PDGF-BB, PDGF-AA, CSF2, FLT1, TEK, HGF, VEGFA, IL6, CSF3, FIGF,IL1RN, CCL11, IL1A, TGFA, PGF, PDGF-AB, IL10, and FGF2, in the first andthe second biological samples; and calculating the ratio (second/first)of the concentrations of the protein. A reduced ratio, as compared to acontrol, of the concentration of CCL5, FLT3LG, IL12(p40), EGF, PDGF-BB,PDGF-AA, CSF3, FLT1, TEK, HGF, VEGFA, or IL6, and an increased ratio, ascompared to a control, of the concentration of CSF2, FIGF, IL1RN, CCL11,IL1A, TGFA, PGF, PDGF AB, IL10, or FGF2 is predictive that the subjectwill respond to the therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof. In one embodiment, the sample is obtained about15 days after initiation of the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof. In one embodiment, the sampleis obtained about 29 days or any first day of each treatment cycle (fourweeks per cycle) after initiation of the therapy comprising lenvatinibor a pharmaceutically acceptable salt thereof.

In a fifth aspect, the disclosure provides a method of treating athyroid or kidney cancer, the method including the step of administeringto a subject in need thereof an effective amount of a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof, wherein thesubject has been identified as having a mutation that is associated withresponsiveness to this therapy, and/or expressing a level or having anexpression ratio of a biomarker that is associated with responsivenessto this therapy, and/or, for the case of thyroid cancer, having anexpression ratio of thyroglobulin that is associated with responsivenessto this therapy.

In a sixth aspect, the disclosure provides a method of predictingresponsiveness of a subject having, suspected of having, or at risk ofdeveloping a thyroid or kidney cancer. The method involves assessing themutational status of NRAS and the pre-treatment concentrations of ANGPT2in a biological sample(s) obtained from the subject. In one embodiment,the presence of a mutation in NRAS (e.g., a NRAS mutation listed inTable 1 or 2) and concentrations of ANGPT2 when entered into thefollowing prediction formula:(0.000751)*(Ang2)+(2.69)*D(NRAS,WT)−(3.92)<0.716, where function D isdefined in detailed description section, that satisfy the formula, areeven more strongly predictive of the subject being responsive to atherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate) than a subject having either ofthese biomarkers individually (slopes, insertions and cut-off value inthe formula can be differently optimized when a different population ofthe sample is analyzed.).

In a seventh aspect, the disclosure provides a method of predictingresponsiveness of a subject having, suspected of having, or at risk ofdeveloping a thyroid or kidney cancer. The method involves assessing themutational status of NRAS or KRAS and the pre-treatment concentrationsof ANGPT2 in a biological sample(s) obtained from the subject. In oneembodiment, the presence of a mutation in NRAS or KRAS (e.g., a NRASmutation or a KRAS mutation listed in Table 1 or 2) and concentrationsof ANGPT2 when entered into the following prediction formula:(0.000869)*(ANG290)+(2.16)*D(KRASNRAS,WT)−(2.24)<0.508, that satisfy theformula, are even more strongly predictive of the subject beingresponsive to a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate) than a subjecthaving either of these biomarkers individually (slopes, insertions andcut-off value in the formula can be differently optimized when adifferent population of the sample is analyzed.).

The following embodiments are envisaged for all of the above aspects. Inone embodiment the lenvatinib or a pharmaceutically acceptable saltthereof is lenvatinib mesylate. In one embodiment, the thyroid cancer isa differentiated thyroid cancer. In another embodiment, the thyroidcancer is a medullary thyroid cancer. In one embodiment, the thyroidcancer is a papillary thyroid cancer. In another embodiment, the thyroidcancer is a follicular thyroid cancer. In another embodiment, thethyroid cancer is a Hürthle-cell thyroid cancer. In a certainembodiment, the thyroid cancer is an advanced radioiodine-refractorydifferentiated thyroid cancer. In one embodiment, the kidney cancer isrenal cell carcinoma. In certain embodiments, the subject is a human. Insome embodiments, the biological sample is selected from the groupconsisting of a blood sample, circulating tumor cells, circulating DNA,a plasma sample, a serum sample, a urine sample, a thyroid sample, athyroid nodule sample, a kidney sample, and a tumor sample. In someembodiments, the method further includes communicating the test resultsto the subject's health care provider. In certain embodiments, themethod further includes modifying the subject's medical record toindicate that the subject is likely or not likely to respond to atherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof. In specific embodiments, the record is created on a computerreadable medium. In certain embodiments, the method further includesprescribing a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof for the subject if the biomarker expressionprofile is predictive that the subject will respond to a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof. Insome embodiments, the method further includes administering to thesubject a therapy comprising lenvatinib or a pharmaceutically acceptablesalt thereof. In some embodiments, the method further comprisesselecting a subject having, or at risk of developing, a cancer thatwould benefit from treatment comprising lenvatinib or a pharmaceuticallyacceptable salt thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the exemplary methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentapplication, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of progression free survival (PFS) ofpatients with mutations in NRAS alone or with mutations in either NRASor KRAS. Log rank test analysis demonstrated that patients withmutations in NRAS alone, or with mutations in either NRAS or KRAS hadbetter PFS than those patients who were wild type for NRAS or KRAS.

FIG. 2 is a schematic diagram of the change in thyroglobulin levelsafter E7080 treatment. “PR” refers to median values among groups ofpatients showing partial response to E7080 therapy. “Others” refers tomedian values among groups of patients having stable disease orprogressive disease after E7080 therapy.

DETAILED DESCRIPTION

This disclosure provides methods and compositions for predicting theresponse of a thyroid or kidney cancer subject (such as a human patient)to a therapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate). The disclosure provides predictivebiomarkers (e.g., protein expression levels and/or gene mutations) toidentify those subjects having, suspected of having, or at risk ofdeveloping, thyroid (e.g., differentiated thyroid cancer) or kidneycancer (e.g., renal cell carcinoma), for whom administering a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate) is likely to be effective or ineffective. Inaddition, the disclosure provides biomarkers that are useful toevaluate/assess continued treatment of thyroid or kidney cancer subjectswith a therapy comprising lenvatinib or a pharmaceutically acceptablesalt thereof (e.g., lenvatinib mesylate). The biomarkers, compositions,and methods described herein are useful in selecting appropriatetherapeutic modalities (e.g., a lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate) therapy) forsubjects suffering from thyroid cancer or kidney cancer. Furthermore,this application provides methods of selecting patients having,suspected of having, or at risk of developing, thyroid or kidney cancerthat could benefit from a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) aswell as methods of treatment.

Definitions

The term “circulating tumor cells” (CTCs) refers to cells that havedetached from a primary tumor and circulate in the bloodstream. CTCs mayconstitute seeds for subsequent growth of additional tumors (metastasis)in different tissues (Kitago et al., Clin. Chem., 55(4):757:764 (2009)).

The term “circulating DNA” refers to DNA that is present in increasedamounts in plasma or serum of cancer patients. Cancer patients havehigher levels of circulating DNA than healthy controls (Leon et al.,Cancer Res., 37: 646-650 (1977); Chuang et al., Head & Neck, 229-234(2010)).

The term “decreased/reduced expression level” means an expression levelthat is lower than the expression level in a control.

The term “elevated expression level” means an expression level that ishigher than the expression level in a control.

The term “lenvatinib” refers to

4-(3-chloro-4(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide.

This compound is disclosed in Example 368 (see, column 270) of U.S. Pat.No. 7,253,286. U.S. Pat. No. 7,253,286 is incorporated by reference inits entirety herein. Lenvatinib mesylate is also referred to as E7080.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein, and refer to both RNA and DNA, including cDNA, genomic DNA,synthetic DNA, and DNA (or RNA) containing nucleic acid analogs.Polynucleotides can have any three-dimensional structure. A nucleic acidcan be double-stranded or single-stranded (i.e., a sense strand or anantisense strand). Non-limiting examples of polynucleotides includegenes, gene fragments, exons, introns, messenger RNA (mRNA), transferRNA, ribosomal RNA, siRNA, micro-RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers, as well as nucleic acid analogs.

The term “pharmaceutically acceptable salt” is not particularlyrestricted as to the type of salt. Examples of such salts include, butare not limited to, inorganic acid addition salt such as hydrochloricacid salt, sulfuric acid salt, carbonic acid salt, bicarnobate salt,hydrobromic acid salt and hydriodic acid salt; organic carboxylic acidaddition salt such as acetic acid salt, maleic acid salt, lactic acidsalt, tartaric acid salt and trifluoroacetic acid salt; organic sulfonicacid addition salt such as methanesulfonic acid salt,hydroxymethanesulfonic acid salt, hydroxyethanesulfonic acid salt,benzenesulfonic acid salt, toluenesulfonic acid salt and taurine salt;amine addition salt such as trimethylamine salt, triethylamine salt,pyridine salt, procaine salt, picoline salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt, N-methylglucamine salt,diethanolamine salt, triethanolamine salt,tris(hydroxymethylamino)methane salt and phenethylbenzylamine salt; andamino acid addition salt such as arginine salt, lysine salt, serinesalt, glycine salt, aspartic acid salt and glutamic acid salt. In oneembodiment, the pharmaceutically acceptable salt is a methanesulfonicacid salt (“mesylate”). The methanesulfonic acid salt form (i.e., themesylate) of4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamideis disclosed in U.S. Pat. No. 7,612,208, which is incorporated byreference herein in its entirety.

“Polypeptide” and “protein” are used interchangeably herein and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification. Typically, a polypeptide describedherein is “isolated” when it constitutes at least 60%, by weight, of thetotal protein in a preparation, e.g., 60% of the total protein in asample. In some embodiments, a polypeptide described herein consists ofat least 75%, at least 90%, or at least 99%, by weight, of the totalprotein in a preparation.

The term “responds/responsive to a therapy” means that the subjectadministered with the therapy shows a positive response to the therapyprovided. Non-limiting examples of such a positive response are: adecrease in tumor size, a decrease in metastasis of a tumor, or anincreased period of survival after treatment.

The term “subject” means a mammal, including but not limited to, ahuman, a chimpanzee, an orangutan, a gorilla, a baboon, a monkey, amouse, a rat, a pig, a horse, a dog, and a cow.

Mutations Associated with Responsiveness to Therapy ComprisingLenvatinib or a Pharmaceutically Acceptable Salt Thereof

Mutations in certain genes such as NRAS, KRAS, VHL, or BRAF arepredictive of the responsiveness of a subject to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate). Non-limiting examples of such mutations are listedin Tables 1 and 2 in the context of the amino acid sequence of theprotein encoded by the respective genes.

TABLE 1 Mutational Biomarkers Gene Mutation BRAF V600E BRAF V600K BRAFK601E BRAF V600R BRAF D594G BRAF V600D BRAF V600M BRAF G469A BRAF V600ABRAF V600G BRAF G466V BRAF G469V BRAF V600_K601>E BRAF L597S BRAF L597VBRAF G464E BRAF G464V BRAF D594N BRAF F595L BRAF L597Q BRAFA598_T599insV BRAF G469R BRAF G469S BRAF L597R BRAF G466E BRAF G469EBRAF Y472S BRAF T599I BRAF K601N BRAF K601del BRAF A598V BRAFT599_R603>I BRAF T599_V600>IAL BRAF Q612* KRAS G12D KRAS G12V KRAS G13DKRAS G12C KRAS G12A KRAS G12S KRAS G12R KRAS G13C KRAS Q61H KRAS G13SKRAS Q61L KRAS G13R KRAS Q61R KRAS A146T KRAS G12F KRAS G13V KRAS G13AKRAS Q61K KRAS L19F KRAS Q61P KRAS G13G KRAS Q61E KRAS A146V KRAS V14IKRAS A59T KRAS G12G KRAS G12N KRAS K117N KRAS G10_A11insG KRAS G12L KRASQ22K NRAS Q61R NRAS Q61K NRAS G12D NRAS G13D NRAS Q61L NRAS G12S NRASQ61H NRAS G12C NRAS G13R NRAS G12V NRAS G13V NRAS G12A NRAS G13C NRASQ61P NRAS G13A NRAS G12R NRAS A18T NRAS Q61E NRAS G60E NRAS G13S NRASG12G NRAS G13G NRAS Q61Q NRAS S65C NRAS A11T NRAS T58I NRAS R68T VHLP81S VHL S68* VHL L89H VHL F148fs*11 VHL S65L VHL R161* VHL S80R VHLV130L VHL L158V VHL S72fs*87 VHL S65* VHL L158Q VHL I151S VHL Q96* VHLV62fs*5 VHL E70* VHL L85P VHL S183* VHL G114C VHL H115N VHL L169P VHLF76del VHL A56fs*11 VHL A149fs*25 VHL E160K VHL Q132* VHL Q195* VHLP172fs*30 VHL L153P VHL Y175fs*27 VHL Q164* VHL G144fs*15 VHL L128fs*31VHL V74D VHL Y175* VHL L184P VHL N78K VHL P99fs*60 VHL R167Q VHL I180NVHL W88* VHL Y156fs*3 VHL L135fs*24 VHL Y185fs*17 VHL R167W VHL L118PVHL C77* VHL Y98* VHL L89P VHL L163P VHL H115Y VHL Y175fs*27 VHL R82PVHL L158P VHL N90I VHL T157fs*2 VHL D126G VHL L89R VHL P86H VHLL135fs*24 VHL C162Y VHL F148fs*11 VHL G144fs*14 VHL P61P VHL F136fs*23VHL S168fs*2 VHL D187fs*27 VHL R107fs*52 VHL T133fs*26 VHL W117* VHLR177* VHL Q73* VHL W88R VHL N141fs*3 VHL R161P VHL E189K VHL I151T VHLY98fs*61 VHL V137fs*7 VHL F119L VHL C162R VHL Q164P VHL A149fs*26 VHLG144* VHL L128P VHL S111N VHL G114R VHL S80N VHL V155L VHL N131fs*28 VHLR58fs*9 VHL W117R VHL N78I VHL R108fs*51 VHL P172fs*30 VHL E10G VHL E12KVHL L153fs*6 VHL L101L VHL V87_W88del VHL L128R VHL M1I VHL G39S VHLE134* VHL K171N VHL P138R VHL G114S VHL G104fs*55 VHL G104fs*55 VHLW117* VHL G104fs*55 VHL L163fs*7 VHL I180fs*22 VHL P81fs*78 VHL D121EVHL S139fs*20 VHL N141fs*18 VHL R167fs*3 VHL H115fs*44 VHL S65fs*2 VHLS38F VHL P40S VHL E41V VHL E51Q VHL P95R VHL V62fs*68 VHL N131fs*28 VHLN131fs*27 VHL V137fs*22 VHL S139S VHL P146fs*13 VHL V166G VHLD187_L188del VHL L188Q VHL M1fs*20 VHL T157T VHL S111S VHL W88C VHLD179fs*23 VHL N150fs*9 VHL V155fs*4 VHL N150fs*9 VHL N78S VHL N174fs*28VHL N90fs*69 VHL Y98F VHL T124fs*35 VHL V155fs*4 VHL V166D VHL Y175D VHLN193fs*22 VHL W88R VHL Y98* VHL A122E VHL P146P VHL G104fs*23 VHL D121GVHL C162W VHL R200W VHL T157I VHL P86L VHL V142fs*17 VHL E160* VHL N78HVHL V155M VHL V142fs*17 VHL L101P VHL P154L VHL I151N VHL F136V VHLN131fs*2 VHL P86S VHL S111G VHL I151M VHL Y185* VHL R182R VHL P59fs*8VHL L169L VHL E186* VHL C162F VHL L188P VHL K196fs*18 VHL N131K VHL S68PVHL I109N VHL R113* VHL S65W VHL D121Y VHL E160fs*10 Key to SelectedMutations: V600_K601>E = amino acids VK are replaced by E; A598_T599insV= insertion of V between A598 and T599; K601del = deletion of K601;Q612* = substitution of Q612 to stop codon; and F148fs*11 = frameshiftoccurred at F148 and a stop codon appeared after 11 amino acids.

TABLE 2 Additional Mutational Biomarkers Gene Mutation BRAF D594V BRAFD594G BRAF F468C BRAF F595L BRAF G464R BRAF G464V BRAF G464E BRAF G466RBRAF G469S BRAF G469E BRAF G469A BRAF G469V BRAF G469R BRAF G596R BRAFK601E BRAF K601N BRAF L597Q BRAF L597V BRAF L597S BRAF L597R BRAF T599IBRAF V600E BRAF V600K BRAF V600R BRAF V600L BRAF D587A BRAF D587E BRAFD594E BRAF E586K BRAF F595S BRAF G466V BRAF G469A BRAF I592M BRAF I592VBRAF K601del BRAF N581S BRAF R444W BRAF S605F BRAF S605N BRAFT599_V600insTT BRAF V471F BRAF V600A BRAF V600D BRAF V600M KRAS A59TKRAS G12A KRAS G12C KRAS G12D KRAS G12F KRAS G12R KRAS G12S KRAS G12VKRAS G13V KRAS G13D KRAS Q61E KRAS Q61K KRAS Q61H KRAS Q61L KRAS Q61RKRAS Q61P KRAS A146T KRAS G13A KRAS G13R KRAS L19F KRAS Q22K NRAS A18TNRAS G12C NRAS G12R NRAS G12S NRAS G12V NRAS G12A NRAS G12D NRAS G13CNRAS G13R NRAS G13S NRAS G13V NRAS G13A NRAS G13D NRAS Q61E NRAS Q61KNRAS Q61H NRAS Q61L NRAS Q61P NRAS A18T VHL F148fs*11 VHL L158Q VHL L85PVHL L89H VHL P81S VHL R161* VHL R167W

The presence in a subject of any one or more of the mutations listed inTable 1 and/or Table 2 is predictive that the subject will respond to atherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate). In the interest of brevity, everypossible combination of mutations from Table 1 and Table 2 suitable foruse in the invention is not expressly listed herein. Nevertheless, itshould be understood that every such combination is contemplated and iswithin the scope of the invention. The subject can have a singlemutation (e.g., NRAS Q61P) or multiple mutations in the same gene (e.g.,NRAS G12D and NRAS Q61R) or single mutations in multiple genes (e.g.,BRAF V600E, NRAS Q61R, KRAS G12R, and VHL P81S); or multiple mutationsin multiple genes (e.g., NRAS G12D, NRAS Q61P, KRAS G12R, and KRASQ61R); or a mixture of single mutations in certain genes and multiplemutations in other genes (e.g., BRAF V600E; NRAS Q61P, NRAS G13V; KRASG12R, KRAS Q61R; and VHL P81S). As few as one mutation listed in Table 1or Table 2 is useful in predicting responsiveness to a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate). In certain embodiments, the mutation(s)is/are in NRAS. Non-limiting examples of NRAS mutations that arepredictive of responsiveness to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) areNRAS Q61P and NRAS Q61R. In other embodiments, the mutation(s) is/are inNRAS and/or KRAS. Non-limiting examples of KRAS mutations that arepredictive of responsiveness to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) areKRAS G12R and KRAS Q61R. In other embodiments, the mutation(s) is/are inNRAS and/or KRAS and/or VHL. A non-limiting example of a VHL mutationthat is predictive of responsiveness to a therapy comprising lenvatinibor a pharmaceutically acceptable salt thereof (e.g., lenvatinibmesylate) is P81S. In yet other embodiments, the mutation(s) is/are inNRAS and/or KRAS and/or BRAF. A non-limiting example of a BRAF mutationthat is predictive of responsiveness to a therapy comprising lenvatinibor a pharmaceutically acceptable salt thereof (e.g., lenvatinibmesylate) is BRAF V600E. In another embodiment, the mutation(s) is/arein NRAS and/or KRAS and/or BRAF and/or VHL.

One or more mutations in genes other than, or in addition to, NRAS,KRAS, VHL and/or BRAF can also be predictive of responsiveness to atherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate). Non-limiting examples of such genesinclude ERBB2, PTEN and MET. Non-limiting examples of mutations in thesegenes that can be predictive of responsiveness to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) include ERBB2 S779_P780insVGS, PTEN N323fs*2 andMET T1010I.T992I.

In one embodiment, the subject has, is suspected of having, or is atrisk of developing a thyroid cancer (e.g., differentiated thyroid cancersuch as papillary or follicular thyroid cancer). In another embodiment,the subject has, is suspected of having, or is at risk of developing akidney cancer (e.g., renal cell carcinoma).

Nucleic acid isolated from biological samples obtained from the subjectcan be analyzed for the presence of one or more of the mutations listedin Table 1 and/or Table 2. Methods of identifying mutations in a nucleicacid are well known in the art.

One method of assessing whether a subject has a mutation in any of thegenes of interest is the method described in Example 1, specifically,the use of Sequenom's OncoCarta™ mutation panels. Other non-limitingmethods for determining if a gene or nucleic acid of interest contains amutation include: Sanger sequencing (chain-termination method),massively parallel signature sequencing (MPSS), Polony sequencing, 454pyrosequencing, Illumina sequencing, SOLiD sequencing, ion semiconductorsequencing, DNA nanoball sequencing, and the simultaneous multiplemutation detection (SMMD) system utilizing an electrochemical array chipand ferrocenyl-naphthalene diimide (FND) (see, Wakai et al., Nucl.Acids. Res., 32(18): e141 (2004).

The proteins of interest can also be isolated from the biologicalsamples from the subject and analyzed for the presence of mutations suchas those disclosed above. Methods of protein sequencing are well knownin the art. Non-limiting examples of such methods include massspectrometry and the Edman degradation reaction. Protein sequencing canbe carried out in both the form of whole-protein analysis or analysis ofenzymatically produced peptides by mass spectrometry (see, Chait,Science. 314(5796):65-6 (2006)). Tandem mass spectrometry (MS/MS), suchas collision-induced dissociation (CID) (4), is a key technique forprotein or peptide sequencing. In this method, gas-phase peptide/proteinions which are generated by ion source are internally heated by multiplecollisions with rare gas atoms. This leads to peptide backbonefragmentation of the C—N bond resulting in a generation of series offragment ions. The sequence information can be read from the series offragment ions.

The biological samples that are used to obtain the nucleic acid orprotein for analysis include, but are not limited to, a blood sample, aplasma sample, a serum sample, circulating tumor cells, circulating DNA,a urine sample, a thyroid tissue sample, a thyroid nodule sample, arenal tissue sample, or a tumor sample.

Thyroglobulin as a Biomarker for Responsiveness to Therapy ComprisingLenvatinib or a Pharmaceutically Acceptable Salt Thereof

In addition to the mutation biomarkers described above, thyroglobulincan also be used as an effective biomarker. Thyroglobulin is the majorprotein found in the thyroid colloid and is central to thyroidphysiology, functioning both as a pro-hormone and a storage site forthyroid hormones. The expression level of thyroglobulin can be used todetermine whether a subject (e.g., one having, suspected of having, orat risk of developing differentiated thyroid cancer) will be more likelyor less likely to respond to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate). Inaddition, the expression level of thyroglobulin can also be used toassess or evaluate whether a subject already being administered atherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate) should continue or terminate thetherapy.

To assess whether a subject will respond effectively to a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof orto evaluate continued treatment with this therapy the following methodcan be employed. A biological sample (e.g., blood, serum, or plasmasample) is obtained from the subject both prior to and afteradministration of lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate). The ratio of the expression levelof thyroglobulin in the two samples (concentration of thyroglobulinafter administration of lenvatinib or a pharmaceutically acceptable saltthereof/concentration of thyroglobulin before administration oflenvatinib or a pharmaceutically acceptable salt thereof) is calculated.If the ratio of the samples from the test subject is less than thecontrol, the subject is determined to be likely to respond to lenvatinibor a pharmaceutically acceptable salt thereof (e.g., lenvatinibmesylate), whereas if the ratio of the samples from the test subject isgreater than or about the same as (at least 90% but less than 100% of)that of the control, the subject is determined to be less likely torespond to lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate). If the subject is predicted to respond totreatment, the therapy with lenvatinib or a pharmaceutically acceptablesalt thereof is recommended to be continued. In the context of the aboveassay, the term “control” means samples obtained pre- and post-treatmentwith lenvatinib or a pharmaceutically acceptable salt thereof from thesame source (e.g., blood, serum or plasma sample) as that of the testsamples and that are taken at the same, or substantially the same, timepoints from a control subject(s) as the test samples, from a subject (orsubjects) who has not responded to treatment with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).The term “control” includes samples obtained in the past (pre- andpost-treatment with the therapy) and used as a reference for futurecomparisons to test samples taken from subjects for which therapeuticresponsiveness is to be predicted. For example, the “control” may bepre-established by an analysis of thyroglobulin expression pre- andpost-treatment with lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate) in one or more subjects that havenot responded to treatment with lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate). Thispre-established reference ratio (which may be an average or median ratiotaken from multiple subjects that have not responded to the therapy) maythen be used for the “control” ratio in the comparison with the testsample.

The “control” may alternatively be pre-established by an analysis ofthyroglobulin expression in one or more subjects that have responded totreatment with lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate). This pre-established reference ratio (whichmay be an average or median ratio taken from multiple subjects that haveresponded to the therapy) may then be used as the “control” ratio in thecomparison with the test sample. In such a comparison, the subject ispredicted to respond to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) ifthe ratio of thyroglobulin levels is comparable to or lower than, forexample is lower than, the same as, or about the same as (at least 90%but less than 100% of), the pre-established reference ratio.

In the above method, the first biological sample can be taken at anytime point prior to treatment with the therapy lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).For example, the first biological sample may be taken minutes, hours,days, weeks, or months before initiation of the therapy, orsubstantially at the same time as the initiation of the therapy. Thesecond biological sample can also be taken from the subject at any timepoint after initiation of treatment with the therapy lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).For example, the second biological sample can be taken minutes, hours,days, weeks, or months after treatment with the therapy lenvatinib or apharmaceutically acceptable salt thereof. Non-limiting examples of thetime points when the second biological sample is taken include: 1 weekto 9 months, 2 weeks to 9 months, 3 weeks to 9 months after, 4 weeks to9 months after, 1 day to 2 weeks after, 2 days to 2 weeks after, 3 daysto 2 weeks after, 4 days to 2 weeks after, 5 days to 2 weeks after, 6days to 2 weeks after, 1 week to 2 weeks after, 1 week to 3 weeks after,1 week to 4 weeks after, and 1 week to 5 weeks after, initiation oftreatment with the therapy lenvatinib or a pharmaceutically acceptablesalt thereof.

The thyroglobulin levels can be determined either by measuring thelevels of mRNA or protein levels. Methods of measuring mRNA and proteinlevels are well known in the art (see, e.g., Sambrook J, Fritsch E F,Maniatis T, eds. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed.(Woodbury, N.Y.: Cold Spring Harbor Laboratory Press; Real-time PCRapplications guide. Bio-Rad Bulletin 5279 (catalog #170-9799)).

In certain embodiments, a subject is determined to respond to lenvatinibor a pharmaceutically acceptable salt thereof (e.g., lenvatinibmesylate), if the subject shows a partial response post treatment withthe therapy. “Partial Response” means at least 30% decrease in the sumof the longest diameter (LD) of target lesions, taking as reference thebaseline summed LD. In some embodiments, a subject is determined torespond to lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate), if the subject shows tumor shrinkagepost-treatment with the therapy. “Tumor shrinkage” (TS) means percentchange of sum of diameters of target lesions, taking as reference thebaseline sum diameters. In other embodiments, a subject is determined torespond to lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate), if the subject shows progression freesurvival. “Progression Free Survival” (PFS) refers to the period fromstart date of treatment to the last date before entering ProgressiveDisease (PD) status. PD means at least 20% increase in the sum of the LDof target lesions, taking as reference the smallest summed LD recordedsince the treatment started, or the appearance of one or more newlesions.

A larger decrease in thyroglobulin levels post-treatment frompre-treatment levels compared to a control (e.g., pre- andpost-treatment samples obtained from a subject who is not responsive toa therapy comprising lenvatinib or a pharmaceutically acceptable saltthereof is predictive of a partial response to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) in differentiated thyroid cancer patients.

A decrease in thyroglobulin levels about 28 days (e.g., 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34 days) after treatment with a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate) is predictive of progression free survivalin differentiated thyroid cancer patients.

A decrease in thyroglobulin levels about 56 days after, about 84 daysafter, about 112 days after, and about 140 days after treatment with atherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate) is predictive of tumor shrinkage indifferentiated thyroid cancer patients.

Cytokine, Chemokine, and Angiogenic Factors as Biomarkers forResponsiveness to Therapy Comprising Lenvatinib or a PharmaceuticallyAcceptable Salt Thereof

A number of genes have been identified whose expression levels (e.g.,mRNA or protein expression levels) are useful in predictingresponsiveness of a subject to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).These genes as identified by Gene ID, related URL, protein ID andUniProtKB Accession Nos. are listed in Table 3.

TABLE 3 List of Blood Biomarkers Official Gene UniProtKB Gene Symbol IDURL Alternative Symbol* Accession No. ANGPT1 284www.ncbi.nlm.nih.gov/gene/284 Ang-1/Ang1 Q5HYA0 ANGPT2 285www.ncbi.nlm.nih.gov/gene/285 Ang-2/ANG-2/Ang2/ANG2 O15123 (90)/ANG290CCL11 6356 www.ncbi.nlm.nih.gov/gene/6356 Eotaxin P51671 FGF2 2247www.ncbi.nlm.nih.gov/gene/2247 FGF-2/FGF2(79)/FGF279 P09038 FGF23 8074www.ncbi.nlm.nih.gov/gene/8074 FGF-23 Q9GZV9 FGF4 2249www.ncbi.nlm.nih.gov/gene/2249 FGF4 (75)/FGF475 P08620 CSF2 1437www.ncbi.nlm.nih.gov/gene/1437 GM-CSF/GMCSF P04141 IFNG 3458www.ncbi.nlm.nih.gov/gene/3458 IFN-g/IFN-γ P01579 IL10 3586www.ncbi.nlm.nih.gov/gene/3586 IL-10 P22301 IL12A (p35) 3592www.ncbi.nlm.nih.gov/gene/3592 IL12A P29459 IL12B (p40) 3593www.ncbi.nlm.nih.gov/gene/3593 IL12B/IL12(p40)/IL-12(p40) P29460 IL133596 www.ncbi.nlm.nih.gov/gene/3596 IL-13 P35225 IL17A 3605www.ncbi.nlm.nih.gov/gene/3605 IL-17 Q16552 IL1A 3552www.ncbi.nlm.nih.gov/gene/3552 IL-1a/IL-1α P01583 IL1B 3553www.ncbi.nlm.nih.gov/gene/3553 IL-1b/IL-1β P01584 IL2 3558www.ncbi.nlm.nih.gov/gene/3558 IL-2 P60568 IL5 3567www.ncbi.nlm.nih.gov/gene/3567 IL-5 P05113 IL6 3569www.ncbi.nlm.nih.gov/gene/3569 IL-6 P05231 IL8 3576www.ncbi.nlm.nih.gov/gene/3576 IL-8 P10145 CXCL10 3627www.ncbi.nlm.nih.gov/gene/3627 IP-10 P02778 CCL2 6347www.ncbi.nlm.nih.gov/gene/6347 MCP-1 P13500 CCL3 6348www.ncbi.nlm.nih.gov/gene/6348 MIP-1a/MIP1a/MIP-1α P10147 CCL4 6351www.ncbi.nlm.nih.gov/gene/6351 MIP-1b/MIP1b/MIP-1β P13236 CCL5 6352www.ncbi.nlm.nih.gov/gene/6352 RANTES P13501 CD40LG 959www.ncbi.nlm.nih.gov/gene/959 sCD40L P29965 CXCL12 6387www.ncbi.nlm.nih.gov/gene/6387 SDF-1a/SDF1a P48061 KDR 3791www.ncbi.nlm.nih.gov/gene/3791 sVEGFR2 P35968 TEK 7010www.ncbi.nlm.nih.gov/gene/7010 Tie-2 Q02763 TNF 7124www.ncbi.nlm.nih.gov/gene/7124 TNFa/TNF-α P01375 FIGF 2277www.ncbi.nlm.nih.gov/gene/2277 VEGFD (78) O43915 EGF 1950www.ncbi.nlm.nih.gov/gene/1950 EGF (80) P01133 FLT3LG 2323www.ncbi.nlm.nih.gov/gene/2323 FLT3 LG (89) P49771 CSF3 1440www.ncbi.nlm.nih.gov/gene/1440 G-CSF/GCSF P09919 HGF 3082www.ncbi.nlm.nih.gov/gene/3082 HGF (86) P14210 IL15 3600www.ncbi.nlm.nih.gov/gene/3600 IL-15 P40933 IL1RN 3557www.ncbi.nlm.nih.gov/gene/3557 IL-1ra P18510 IL4 3565www.ncbi.nlm.nih.gov/gene/3565 IL-4 P05112 IL7 3574www.ncbi.nlm.nih.gov/gene/3574 IL-7 P13232 PDGFA 5154www.ncbi.nlm.nih.gov/gene/5154 PDGFA P04085 PDGFB 5155www.ncbi.nlm.nih.gov/gene/5155 PDGFB P01127 PGF 5228www.ncbi.nlm.nih.gov/gene/5228 PGF (91) P49763 FLT1 2321www.ncbi.nlm.nih.gov/gene/2321 sVEGFR1 P17948 FLT4 2324www.ncbi.nlm.nih.gov/gene/2324 sVEGFR3 P35916 TGFA 7039www.ncbi.nlm.nih.gov/gene/7039 TGFa/TGF-α P01135 VEGFA 7422www.ncbi.nlm.nih.gov/gene/7422 VEGF/VEGFA P15692 (100)/VEGFA100 *Otheralternate symbols PDGF-AA (alternative symbol PDGFAA); homo dimer ofPDGFA PDGF-AB (alternative symbol PDGFAB); hetero dimer of PDGFA andPDGFB PDGF-BB (alternative symbol PDGFBB); homo dimer of PDGFB IL-12p70;hetero dimer of IL12A(p35) and IL12B(p40)

A low expression (e.g., mRNA or protein expression) level (compared to acontrol) of certain genes listed in Table 3 is indicative/predictivethat a subject will respond to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).For example, low concentrations (compared to a control) of ANGPT2,VEGFA, IFNG, and KDR in a biological sample obtained from a subjectprior to treatment with the therapy are predictive that a given subjectwill respond to a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate). In this context,the term “control” includes a sample (from the same tissue) obtainedfrom a subject who is known to not respond to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate). The term “control” also includes a sample obtainedin the past and used as a reference for future comparisons to testsamples taken from subjects for which therapeutic responsiveness is tobe predicted. For example, the “control” expression level for aparticular gene in a particular cell type or tissue may bepre-established by an analysis of gene expression in one or moresubjects that have not responded to treatment with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).This pre-established reference value (which may be an average or medianexpression level taken from multiple subjects that have responded to thetherapy) may then be used for the “control” expression level in thecomparison with the test sample. The “control” expression level for aparticular gene in a particular cell type or tissue may alternatively bepre-established by an analysis of gene expression in one or moresubjects that have responded to treatment with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).This pre-established reference value (which may be an average or medianexpression level taken from multiple subjects that have responded to thetherapy) may then be used as the “control” expression level in thecomparison with the test sample. In such a comparison, the subject ispredicted to respond to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) ifthe expression level of the gene being analyzed is comparable to orlower than, for example is lower than, the same as, or about the same as(at least 85% but less than 100% of, the pre-established reference.

A high expression (e.g., mRNA or protein expression) level (compared toa control) of certain genes listed in Table 3 is predictive that asubject will respond to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).For example, high concentrations (compared to a control) of PDGF-AB,FGF2, CSF3, IL6, IL13, FLT4, CCL3, and CCL4 in a biological sampleobtained from a subject prior to treatment with the therapy arepredictive that a given subject will respond to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate). In this context, the term “control” is identicalto that described in the paragraph above, except that when the “control”expression level for a particular gene in a particular cell type ortissue is alternatively pre-established by an analysis of geneexpression in one or more subjects that have responded to treatment withlenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate), the subject is predicted to respond to a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate) if the expression level of the gene beinganalyzed is comparable to or higher than, for example is higher than,the same as, or about the same as (at least 85% but less than 100% of,the pre-established reference.

It is also envisaged that subjects be administered with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) forshort periods of time to determine whether the administered therapy willbe effective for the subject. The determination of effectiveness of thetherapy is made based on the expression (e.g., mRNA or proteinexpression) levels of certain genes in biological samples obtained fromthese subjects at different time points post-treatment. Based on theexpression levels of these genes, one can predict whether the subjectwill respond to continued treatment. Thus, these methods are useful inassessing or evaluating whether it is advisable to continueadministration of lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate). For example, low concentrations(compared to a control) of certain genes, e.g., ANGPT2 and/or IL13 about5 days to about 18 days after initiation of therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) are predictive that the subject will have abeneficial clinical outcome (e.g., tumor response and/or tumorshrinkage) upon continued therapy with lenvatinib compounds. Similarly,high concentrations (compared to a control) of certain genes, e.g., IL10and/or CXCL12 about 5 days to about 18 days after initiation of therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate) are also predictive that the subject willhave a beneficial clinical outcome (e.g., tumor response and/or tumorshrinkage) upon continued therapy with lenvatinib compounds.

In addition, low concentrations (compared to a control) of certaingenes, e.g., VEGFA, IL6, and/or PGF about 5 weeks (e.g., 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks) after initiation of therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) are predictive that the subject will have abeneficial clinical outcome (e.g., tumor response and/or tumorshrinkage) upon continued therapy with lenvatinib compounds. Also, highconcentrations (compared to a control) of certain genes, e.g., CCL5about 5 weeks (e.g., 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks) afterinitiation of therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate) are predictive thatthe subject will have a beneficial clinical outcome (e.g., tumorresponse and/or tumor shrinkage) upon continued therapy with lenvatinibcompounds.

The ratio of the expression (e.g., mRNA or protein expression) level ofcertain genes post-treatment over pre-treatment with lenvantib or apharmaceutically acceptable salt thereof (compared to a control) canalso be useful in predicting whether a subject will have a beneficialclinical outcome (e.g., best overall response, tumor shrinkage,progression free survival) upon continued therapy with lenvatinibcompounds. For example, a reduced ratio, as compared to a control, ofthe expression level of certain genes, e.g., CCL5, FLT3LG, IL12(p40),EGF, PDGF-BB, PDGF-AA, CSF3, FLT1, TEK, HGF, VEGFA, or IL6 is indicativethat the subject will respond to the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof. In addition, an increasedratio, as compared to a control, of the concentration of certain genes,e.g., CSF2, FIGF, IL1RN, CCL11, IL1A, TGFA, PGF, PDGF AB, IL10, or FGF2is predictive that the subject will respond to the therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof.

A reduced ratio (compared to a control) of the expression level ofcertain genes, e.g., CCL5, FLT3LG based on expression about 5 days toabout 18 days after initiation of therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) andthe expression level of the same gene prior to initiation of thistherapy is indicative that the subject will have progression freesurvival.

An increased ratio (compared to a control) of the expression level ofcertain genes, e.g., CSF3 or FGF2 based on expression about 5 days toabout 18 days after initiation of therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) andthe expression level of the same gene prior to initiation of thistherapy is indicative that the subject will have tumor response.

An increased ratio (compared to a control) of the expression level ofcertain genes, e.g., CSF3, IL10 or FGF2 based on expression about 5 daysto about 18 days after initiation of therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) andthe expression level of the same gene prior to initiation of thistherapy is indicative that the subject will show tumor shrinkage.

An increased ratio (compared to a control) of the expression level ofcertain genes, e.g., FIGF, ILIRN, PDGFAB or IL10 based on expressionabout 5 days to about 18 days after initiation of therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) and the expression level of the same gene prior toinitiation of this therapy is indicative that the subject will haveprogression free survival.

A reduced ratio of the expression level of certain genes, e.g., FLT3LG,IL12, EGF, PDGFBB, PDGFAA, CSF3, or FLT1 based on expression about 5weeks (e.g., 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks) afterinitiation of therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate) and the expressionlevel of the same gene prior to initiation of this therapy is indicativethat the subject will have progression free survival.

An increased ratio of the expression level of certain genes, e.g., CCL11based on expression about 5 weeks (e.g., 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks) after initiation of therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) andthe expression level of the same gene prior to initiation of thistherapy is indicative that the subject will exhibit tumor response.

An increased ratio of the expression level of certain genes, e.g., IL1Aor TGFA based on expression about 5 weeks (e.g., 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks) after initiation of therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) and the expression level of the same gene prior toinitiation of this therapy is predictive that the subject will exhibittumor shrinkage.

A reduced ratio of the expression level of certain genes, e.g., FLT1,TEK, VEGFA, or IL6 based on expression about 5 weeks (e.g., 3 weeks, 4weeks, 5 weeks, 6 weeks, 7 weeks) after initiation of therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) and the expression level of the same gene about 5days to about 18 days after initiation of this therapy is indicativethat the subject will exhibit tumor shrinkage.

A reduced ratio of the expression level of certain genes, e.g., TEK,HGF, or VEGFA based on expression about 5 weeks (e.g., 3 weeks, 4 weeks,5 weeks, 6 weeks, 7 weeks) after initiation of therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) and the expression level of the same gene about 5days to about 18 days after initiation of this therapy is predictivethat the subject will show the best overall response.

An increased ratio of the expression level of certain genes, e.g., PGFbased on expression about 5 weeks (e.g., 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks) after initiation of therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) andthe expression level of the same gene about 5 days to about 18 daysafter initiation of this therapy is indicative that the subject willexhibit tumor shrinkage, whereas an increased ratio under the sameconditions of e.g., FGF2 is predictive of the subject exhibiting a tumorresponse.

The progression free survival observed above can be, for example, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 13 months, 14 months, 15 months, 16 months, 17months, 18 months, 19 months, 20 months, 21 months, 22 months, 23months, or 24 months, or about 4 months, about 5 months, about 6 months,about 7 months, about 8 months, about 9 months, about 10 months, about11 months, about 12 months, about 13 months, about 14 months, about 15months, about 15 months, about 17 months, about 18 months, about 19months, about 20 months, about 21 months, about 22 months, about 23months, or about 24 months.

In determining whether the ratio is increased or decreased comparison ismade to a control. In this context, the term “control” includes samplesobtained from the same source (e.g., blood, serum or plasma sample) asthat of the test samples and that are taken at the same, orsubstantially the same, time points as the test samples, from a subject(or subjects) who has not responded to treatment with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).The term “control” includes samples obtained in the past (pre- andpost-treatment with the therapy) and used as a reference for futurecomparisons to test samples taken from subjects for which therapeuticresponsiveness is to be predicted. For example, the “control” may be apre-established ratio of the expression of the gene of interest pre- andpost-treatment with lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate) in one or more subjects that havenot responded to treatment with lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate). Thispre-established reference ratio (which may be an average or median ratiotaken from multiple subjects that have not responded to the therapy) maythen be used for the “control” ratio in the comparison with the testsample.

The “control” may alternatively be pre-established by an analysis ofexpression of the gene of interest in one or more subjects who haveresponded to treatment with lenvatinib or a pharmaceutically acceptablesalt thereof (e.g., lenvatinib mesylate). This pre-established referenceratio (which may be an average or median ratio taken from multiplesubjects that have responded to the therapy) may then be used as the“control” ratio in the comparison with the test sample. In such acomparison, the subject is predicted to respond to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) if the ratio of expression levels of the gene iscomparable to, for example, the same as or about the same as (at least90% but less than 100% of), the pre-established reference ratio.

Combinatorial Methods

Any of the above biomarkers may be assessed in combination to determinewhether a subject will respond to, or benefit from continued,administration of a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate). For example, anyone or more of the mutation biomarkers may be assessed in combinationwith thyroglobulin expression ratios and/or expression levels orexpression ratios of cytokine, chemokine, or angiogenic factors, and/orhistological analysis. In some instances, a mutational biomarker(s) isassessed in combination with histological analysis. In other cases, amutational biomarker(s) is assessed in combination with thyroglobulinexpression ratios. In some instances, a mutational biomarker(s) isassessed in combination with expression levels or expression ratios ofone or more cytokine, chemokine, or angiogenic factors. In oneembodiment, the mutational status of NRAS is assessed in the biologicalsample obtained from the subject and considered in combination withpre-treatment concentrations of ANGPT2. In another embodiment, themutational status of NRAS or KRAS is assessed in the biological sampleobtained from the subject and considered in combination withpre-treatment concentrations of ANGPT2. Such combinatorial biomarkeranalyses provide even stronger predictive value than studying individualbiomarkers, and are useful, for example, in predicting responsiveness ofa subject to a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate).

Statistical analysis can be used to determine which markers when used incombination are better associated with a desired clinical outcome thanthe individual markers. A non-limiting example of such an analysis isprovided in Example 4 of this application.

The combination of the expression levels of VEGFA and ANGPT2 prior toinitiation of a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate) (“pre-treatment”)can be a better predictor of response to the therapy than each of theseindividual blood biomarkers. For example, if the pre-treatmentconcentrations of VEGFA and ANGPT2 in a subject when entered into thefollowing prediction formula:(0.000261)*(Ang2)+(0.00126)*(VEGFA100)−(1.09)<−0.24render this formula true (i.e. if the value is <−0.24 (e.g., −1.0)),then the subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) after taking the therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) than subjects whose concentrations of these factorsdo not satisfy the formula (slopes, insertions and cut-off value in theformula can be differently optimized when different population of thesample is analyzed.). For another example, if the pre-treatmentconcentrations of VEGFA, ANGPT2, and GCSF in a subject when entered intothe following prediction formula:(0.000591)*(ANG290)+(−0.0178)*(GCSF)+(0.00142)*(VEGFA100)−(−0.671)<0.651render this formula true (i.e. if the value is <0.651), then the subjectis predicted to have a stronger clinical outcome (e.g., progression freesurvival) after taking the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects whose pre-treatment concentrations of these factors do notsatisfy the formula (slopes, insertions and cut-off value in the formulacan be differently optimized when different population of the sample isanalyzed.).

For further example, if the pre-treatment concentrations of IL13 andMIP1a in a subject when entered into the following prediction formula:(−0.0459)*(IL13)+(0.0459)*(MIP1a)−(0.0395)<0.268render this formula true (i.e. if the value is <0.268), then the subjectis predicted to have a stronger clinical outcome (e.g., progression freesurvival) after taking the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects whose concentrations of these factors do not satisfy theformula (slopes, insertions and cut-off value in the formula can bedifferently optimized when different population of the sample isanalyzed.).

For further example, if the pre-treatment concentrations IL13, MIP1a,and MIP1b in a subject when entered into the following predictionformula:(−0.0353)*(IL13)+(0.0713)*(MIP1a)+(−0.0154)*(MIP1b)−(0.188)<0.222render this formula true (i.e. if the value is <0.222), then the subjectis predicted to have a stronger clinical outcome (e.g., progression freesurvival) after taking the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects whose pre-treatment concentrations of these factors do notsatisfy the formula (slopes, insertions and cut-off value in the formulacan be differently optimized when different population of the sample isanalyzed.).

The combination of a mutation(s) and the expression levels of VEGFA andMIP1b prior to initiation of a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)(“pre-treatment”) can also be a better predictor of response to thetherapy than each of these individual mutational and blood biomarkers.For example, if the sample from the subject has a mutation in NRAS(e.g., one of those listed in Table 1 or 2) and the pre-treatmentconcentrations of VEGFA and MIP1b in a subject when entered into thefollowing prediction formula:(−0.025)*(MIP1b)+(−0.00616)*(VEGFA100)+(3.32)*D(NRAS,WT)−(−0.52)<1.81(The function D(g, s) is 1 when mutation status of gene(s) g is statuss, and 0 when g is not s. The status scan be “WT” (wild type) or “MU”(mutation). For the case of multiple-genes, mutation status is “MU” ifone or more genes have mutation and “WT” only for the case that allgenes are wild-type)render this formula true (i.e. if the value is <1.81 (e.g., 1.0)), thenthe subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects who have wild type NRAS and whose pre-treatmentconcentrations of VEGFA and MIP1b do not satisfy the formula (slopes,insertions and cut-off value in the formula can be differently optimizedwhen different population of the sample is analyzed.).

For another example, if the sample from the subject has a mutation inNRAS (e.g., one of those listed in Table 1 or 2) and the pre-treatmentconcentrations of VEGFA, MIP1b, and sVEGFR3 in a subject when enteredinto the following prediction formula:(−0.0494)*(MIP1b)+(−0.000472)*(sVEGFR3)+(−0.0119)*(VEGFA100)+(4.66)*D(NRAS,WT)−(−5.9)<3.55render this formula true (i.e. if the value is <3.55 (e.g., 3.0)), thenthe subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects who have wild type NRAS and whose pre-treatmentconcentrations of VEGFA, MIP1b, and sVEGFR3 do not satisfy the formula(slopes, insertions and cut-off value in the formula can be differentlyoptimized when different population of the sample is analyzed.).

For further example, if the sample from the subject has a mutation inNRAS (e.g., one of those listed in Table 1 or 2) and the pre-treatmentconcentrations of VEGFA, MIP1b, sVEGFR3, and Ang2 in a subject whenentered into the following prediction formula:(0.00148)*(Ang2)+(−0.0606)*(MIP1b)+(−0.000917)*(sVEGFR3)+(−0.0177)*(VEGFA100)+(6.58)*D(NRAS,WT)−(−5.78)<3.97render this formula true (i.e. if the value is <3.97 (e.g., 3.0)), thenthe subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects who have wild type NRAS and whose pre-treatmentconcentrations of VEGFA, MIP1b, sVEGFR3, and Ang2 do not satisfy theformula (slopes, insertions and cut-off value in the formula can bedifferently optimized when different population of the sample isanalyzed.).

For further example, if the sample from the subject has a mutation inNRAS (e.g., one of those listed in Table 1 or 2) and the pre-treatmentconcentrations of Ang2 in a subject when entered into the followingprediction formula:(0.000751)*(Ang2)+(2.69)*D(NRAS,WT)−(3.92)<0.716render this formula true (i.e. if the value is <0.716 (e.g., 0.5)), thenthe subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects who have wild type NRAS and whose pre-treatmentconcentrations of Ang2 do not satisfy the formula (slopes, insertionsand cut-off value in the formula can be differently optimized whendifferent population of the sample is analyzed.).

For a further example, if the sample from the subject has a mutation inNRAS (e.g., one of those listed in Table 1 or 2) and the pre-treatmentconcentrations of ANG2(90) in a subject when entered into the followingprediction formula:(0.000972)*(ANG290)+(2.75)*D(NRAS,WT)−(2.96)<0.633render this formula true (i.e. if the value is <0.633 (e.g., 0.5)), thenthe subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects who have wild type NRAS and whose pre-treatmentconcentrations of ANG2(90) do not satisfy the formula (slopes,insertions and cut-off value in the formula can be differently optimizedwhen different population of the sample is analyzed.).

For another example, if the sample from the subject has a mutation inNRAS or KRAS (e.g., one of those listed in Table 1 or 2) and thepre-treatment concentrations of ANG2(90) in a subject when entered intothe following prediction formula:(0.000869)*(ANG290)+(2.16)*D(KRASNRAS,WT)−(2.24)<0.508render this formula true (i.e. if the value is <0.508 (e.g., 0.4)), thenthe subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects who have wild type NRAS or KRAS and whose pre-treatmentconcentrations of ANG2(90) do not satisfy the formula (slopes,insertions and cut-off value in the formula can be differently optimizedwhen different population of the sample is analyzed.).

For another example, if the sample from the subject has a mutation inNRAS, KRAS, or BRAF (e.g., one of those listed in Table 1 or 2) and thepre-treatment concentrations of MIP1a in a subject when entered into thefollowing prediction formula:(−0.0281)*(MIP1a)+(2.19)*D(BRAFKRASNRAS,WT)−(−0.41)<−0.0348render this formula true (i.e. if the value is <−0.0348 (e.g., −1.0)),then the subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects who have wild type NRAS or KRAS or BRAF and whosepre-treatment concentrations of MIP1a do not satisfy the formula(slopes, insertions and cut-off value in the formula can be differentlyoptimized when different population of the sample is analyzed.).

For another example, if the sample from the subject has a mutation inNRAS, KRAS, or BRAF (e.g., one of those listed in Table 1 or 2) and thepre-treatment concentrations of IL6, VEGFA, MIP1a, and MIP1b in asubject when entered into the following prediction formula:(0.126)*(IL6)+(−0.193)*(MIP1a)+(−0.0775)*(MIP1b)+(−0.0514)*(VEGFA100)+(7.94)*D(BRAFKRASNRAS,WT)−(−14.4)<4.69render this formula true (i.e. if the value is <4.69 (e.g., 3.0)), thenthe subject is predicted to have a stronger clinical outcome (e.g.,progression free survival) with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)than subjects who have wild type NRAS or KRAS or BRAF and whosepre-treatment concentrations of IL6, VEGFA, MIP1a, and MIP1b do notsatisfy the formula (slopes, insertions and cut-off value in the formulacan be differently optimized when different population of the sample isanalyzed.).Biological Samples

Suitable biological samples for the methods described herein include anybiological fluid, cell, tissue, or fraction thereof, which includesanalyte biomolecules of interest such as nucleic acid (e.g., DNA ormRNA) or protein. A biological sample can be, for example, a specimenobtained from a subject (e.g., a mammal such as a human) or can bederived from such a subject. For example, a sample can be a tissuesection obtained by biopsy, or cells that are placed in or adapted totissue culture. A biological sample can also be a biological fluid suchas urine, blood, plasma, serum, saliva, semen, sputum, cerebral spinalfluid, tears, or mucus, or such a sample absorbed onto a substrate(e.g., glass, polymer, paper). A biological sample can also include athyroid tissue sample, a renal tissue sample, a tumor sample,circulating tumor cells, and circulating DNA. In specific embodiments,the biological sample is a tumor cell(s) or a cell(s) obtained from aregion of the subject suspected of containing a tumor or a pre-cancerouslesion. For example, the biological sample may be a thyroid tumor sampleor a renal tumor sample. A biological sample can be furtherfractionated, if desired, to a fraction containing particular celltypes. For example, a blood sample can be fractionated into serum orinto fractions containing particular types of blood cells such as redblood cells or white blood cells (leukocytes). If desired, a sample canbe a combination of samples from a subject such as a combination of atissue and fluid sample.

The biological samples can be obtained from a subject, e.g., a subjecthaving, suspected of having, or at risk of developing, a cancer. Incertain embodiments, the subject has a thyroid cancer. In someembodiments, the subject has a differentiated thyroid cancer (e.g.,papillary thyroid cancer, follicular thyroid cancer). In otherembodiments, the subject has a medullary thyroid cancer. In certainembodiments, the subject has a kidney cancer. In some embodiments, thesubject has a renal cell carcinoma. Any suitable methods for obtainingthe biological samples can be employed, although exemplary methodsinclude, e.g., phlebotomy, swab (e.g., buccal swab), or fine needleaspirate biopsy procedure. Non-limiting examples of tissues susceptibleto fine needle aspiration include lymph node, lung, thyroid, breast,skin, and liver. Samples can also be collected, e.g., by microdissection(e.g., laser capture microdissection (LCM) or laser microdissection(LMD)).

Methods for obtaining and/or storing samples that preserve the activityor integrity of molecules (e.g., nucleic acids or proteins) in thesample are well known to those skilled in the art. For example, abiological sample can be further contacted with one or more additionalagents such as appropriate buffers and/or inhibitors, includingnuclease, protease and phosphatase inhibitors, which preserve orminimize changes in the molecules (e.g., nucleic acids or proteins) inthe sample. Such inhibitors include, for example, chelators such asethylenediamine tetraacetic acid (EDTA), ethylene glycolbis(P-aminoethyl ether) N,N,N1,N1-tetraacetic acid (EGTA), proteaseinhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin,leupeptin, antipain and the like, and phosphatase inhibitors such asphosphate, sodium fluoride, vanadate and the like. Appropriate buffersand conditions for isolating molecules are well known to those skilledin the art and can be varied depending, for example, on the type ofmolecule in the sample to be characterized (see, for example, Ausubel etal. Current Protocols in Molecular Biology (Supplement 47), John Wiley &Sons, New York (1999); Harlow and Lane, Antibodies: A Laboratory Manual(Cold Spring Harbor Laboratory Press (1988); Harlow and Lane, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); TietzTextbook of Clinical Chemistry, 3rd ed. Burtis and Ashwood, eds. W.B.Saunders, Philadelphia, (1999)). A sample also can be processed toeliminate or minimize the presence of interfering substances. Forexample, a biological sample can be fractionated or purified to removeone or more materials that are not of interest. Methods of fractionatingor purifying a biological sample include, but are not limited to,chromatographic methods such as liquid chromatography, ion-exchangechromatography, size-exclusion chromatography, or affinitychromatography. For use in the methods described herein, a sample can bein a variety of physical states. For example, a sample can be a liquidor solid, can be dissolved or suspended in a liquid, can be in anemulsion or gel, or can be absorbed onto a material.

Assessing Expression of Biomarkers

Gene expression can be detected as, e.g., protein or mRNA expression ofa target gene. That is, the presence or expression level (amount) of agene can be determined by detecting and/or measuring the level of mRNAor protein expression of the gene. In some embodiments, gene expressioncan be detected as the activity of a protein encoded by a gene such as agene depicted in Table 3.

A variety of suitable methods can be employed to detect and/or measurethe level of mRNA expression of a gene. For example, mRNA expression canbe determined using Northern blot or dot blot analysis, reversetranscriptase-PCR (RT-PCR; e.g., quantitative RT-PCR), in situhybridization (e.g., quantitative in situ hybridization) or nucleic acidarray (e.g., oligonucleotide arrays or gene chips) analysis. Details ofsuch methods are described below and in, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual Second Edition vol. 1, 2 and 3.Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., USA,November 1989; Gibson et al. (1999) Genome Res., 6(10):995-1001; andZhang et al. (2005) Environ. Sci. Technol., 39(8):2777-2785; U.S.Publication No. 2004086915; European Patent No. 0543942; and U.S. Pat.No. 7,101,663; the disclosures of each of which are incorporated hereinby reference in their entirety.

In one example, the presence or amount of one or more discrete mRNApopulations in a biological sample can be determined by isolating totalmRNA from the biological sample (see, e.g., Sambrook et al. (supra) andU.S. Pat. No. 6,812,341) and subjecting the isolated mRNA to agarose gelelectrophoresis to separate the mRNA by size. The size-separated mRNAsare then transferred (e.g., by diffusion) to a solid support such as anitrocellulose membrane. The presence or amount of one or more mRNApopulations in the biological sample can then be determined using one ormore detectably-labeled-polynucleotide probes, complementary to the mRNAsequence of interest, which bind to and thus render detectable theircorresponding mRNA populations. Detectable-labels include, e.g.,fluorescent (e.g., umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride, allophycocyanin (APC), or phycoerythrin), luminescent (e.g.,europium, terbium, Qdot™ nanoparticles supplied by the Quantum DotCorporation, Palo Alto, Calif.), radiological (e.g., 1251, 1311, 35S,32P, 33P, or 3H), and enzymatic (horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase) labels.

In another example, the presence or amount of discrete populations ofmRNA (e.g., mRNA encoded by one or more genes depicted in Table 3) in abiological sample can be determined using nucleic acid (oroligonucleotide) arrays (e.g., an array described below under “Arraysand Kits”). For example, isolated mRNA from a biological sample can beamplified using RT-PCR with, e.g., random hexamer or oligo(dT)-primermediated first strand synthesis. The amplicons can be fragmented intoshorter segments. The RT-PCR step can be used to detectably-label theamplicons, or, optionally, the amplicons can be detectably-labeledsubsequent to the RT-PCR step. For example, the detectable-label can beenzymatically (e.g., by nick-translation or kinase such as T4polynucleotide kinase) or chemically conjugated to the amplicons usingany of a variety of suitable techniques (see, e.g., Sambrook et al.,supra). The detectably-labeled-amplicons are then contacted with aplurality of polynucleotide probe sets, each set containing one or moreof a polynucleotide (e.g., an oligonucleotide) probe specific for (andcapable of binding to) a corresponding amplicon, and where the pluralitycontains many probe sets each corresponding to a different amplicon.Generally, the probe sets are bound to a solid support and the positionof each probe set is predetermined on the solid support. The binding ofa detectably-labeled amplicon to a corresponding probe of a probe setindicates the presence or amount of a target mRNA in the biologicalsample. Additional methods for detecting mRNA expression using nucleicacid arrays are described in, e.g., U.S. Pat. Nos. 5,445,934; 6,027,880;6,057,100; 6,156,501; 6,261,776; and 6,576,424; the disclosures of eachof which are incorporated herein by reference in their entirety.

Methods of detecting and/or for quantifying a detectable label depend onthe nature of the label. The products of reactions catalyzed byappropriate enzymes (where the detectable label is an enzyme; see above)can be, without limitation, fluorescent, luminescent, or radioactive orthey may absorb visible or ultraviolet light. Examples of detectorssuitable for detecting such detectable labels include, withoutlimitation, x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers.

The expression of a gene can also be determined by detecting and/ormeasuring expression of a protein encoded by the gene. Methods ofdetermining protein expression are well known in the art. A generallyused method involves the use of antibodies specific for the targetprotein of interest. For example, methods of determining proteinexpression include, but are not limited to, western blot or dot blotanalysis, immunohistochemistry (e.g., quantitativeimmunohistochemistry), immunocytochemistry, enzyme-linked immunosorbentassay (ELISA), enzyme-linked immunosorbent spot (ELISPOT, Coligan, J.E., et al., eds. (1995) Current Protocols in Immunology. Wiley, NewYork), or antibody array analysis (see, e.g., U.S. Publication Nos.20030013208 and 2004171068, the disclosures of each of which areincorporated herein by reference in their entirety). Further descriptionof many of the methods above and additional methods for detectingprotein expression can be found in, e.g., Sambrook et al. (supra).

In one example, the presence or amount of protein expression of a gene(e.g., a gene depicted in Table 3) can be determined using a westernblotting technique. For example, a lysate can be prepared from abiological sample, or the biological sample itself, can be contactedwith Laemmli buffer and subjected to sodium-dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE-resolvedproteins, separated by size, can then be transferred to a filtermembrane (e.g., nitrocellulose) and subjected to immunoblottingtechniques using a detectably-labeled antibody specific to the proteinof interest. The presence or amount of bound detectably-labeled antibodyindicates the presence or amount of protein in the biological sample.

In another example, an immunoassay can be used for detecting and/ormeasuring the protein expression of a gene (e.g., a gene depicted inTable 3). As above, for the purposes of detection, an immunoassay can beperformed with an antibody that bears a detection moiety (e.g., afluorescent agent or enzyme). Proteins from a biological sample can beconjugated directly to a solid-phase matrix (e.g., a multi-well assayplate, nitrocellulose, agarose, sepharose, encoded particles, ormagnetic beads) or it can be conjugated to a first member of a specificbinding pair (e.g., biotin or streptavidin) that attaches to asolid-phase matrix upon binding to a second member of the specificbinding pair (e.g., streptavidin or biotin). Such attachment to asolid-phase matrix allows the proteins to be purified away from otherinterfering or irrelevant components of the biological sample prior tocontact with the detection antibody and also allows for subsequentwashing of unbound antibody. Here as above, the presence or amount ofbound detectably-labeled antibody indicates the presence or amount ofprotein in the biological sample.

There is no particular restriction as to the form of the antibody andthe present disclosure includes polyclonal antibodies, as well asmonoclonal antibodies. The antiserum obtained by immunizing animals,such as rabbits with a protein of the invention, as well polyclonal andmonoclonal antibodies of all classes, human antibodies, and humanizedantibodies produced by genetic recombination, are also included.

An intact protein or its partial peptide may be used as the antigen forimmunization. As partial peptides of the proteins, for example, theamino (N)-terminal fragment of the protein and the carboxy (C)-terminalfragment can be given.

A gene encoding a protein of interest or a fragment thereof is insertedinto a known expression vector, and, by transforming the host cells withthe vector described herein, the desired protein or a fragment thereofis recovered from outside or inside the host cells using standardmethods. This protein can be used as the sensitizing antigen. Also,cells expressing the protein, cell lysates, or a chemically synthesizedprotein of the invention may be also used as a sensitizing antigen.

The mammal that is immunized by the sensitizing antigen is notrestricted; however, it is preferable to select animals by consideringthe compatibility with the parent cells used in cell fusion. Generally,animals belonging to the orders rodentia, lagomorpha, or primates areused. Examples of animals belonging to the order of rodentia that may beused include, for example, mice, rats, and hamsters. Examples of animalsbelonging to the order of lagomorpha that may be used include, forexample, rabbits. Examples of animals belonging to the order of primatesthat may be used include, for example, monkeys. Examples of monkeys tobe used include the infraorder catarrhini (old world monkeys), forexample, Macaca fascicularis, rhesus monkeys, sacred baboons, andchimpanzees.

Well-known methods may be used to immunize animals with the sensitizingantigen. For example, the sensitizing antigen is injectedintraperitoneally or subcutaneously into mammals. Specifically, thesensitizing antigen is suitably diluted and suspended in physiologicalsaline, phosphate-buffered saline (PBS), and so on, and mixed with asuitable amount of general adjuvant if desired, for example, withFreund's complete adjuvant. Then, the solution is emulsified andinjected into the mammal. Thereafter, the sensitizing antigen suitablymixed with Freund's incomplete adjuvant is preferably given severaltimes every 4 to 21 days. A suitable carrier can also be used whenimmunizing and animal with the sensitizing antigen. After theimmunization, the elevation in the level of serum antibody is detectedby usual methods.

Polyclonal antibodies against the proteins of the present disclosure canbe prepared as follows. After verifying that the desired serum antibodylevel has been reached, blood is withdrawn from the mammal sensitizedwith antigen. Serum is isolated from this blood using conventionalmethods. The serum containing the polyclonal antibody may be used as thepolyclonal antibody, or according to needs, the polyclonalantibody-containing fraction may be further isolated from the serum. Forexample, a fraction of antibodies that specifically recognize theprotein of the invention may be prepared by using an affinity column towhich the protein is coupled. Then, the fraction may be further purifiedby using a Protein A or Protein G column in order to prepareimmunoglobulin G or M.

To obtain monoclonal antibodies, after verifying that the desired serumantibody level has been reached in the mammal sensitized with theabove-described antigen, immunocytes are taken from the mammal and usedfor cell fusion. For this purpose, splenocytes can be mentioned aspreferable immunocytes. As parent cells fused with the aboveimmunocytes, mammalian myeloma cells are preferably used. Morepreferably, myeloma cells that have acquired the feature, which can beused to distinguish fusion cells by agents, are used as the parent cell.

The cell fusion between the above immunocytes and myeloma cells can beconducted according to known methods, for example, the method byMilstein et al. (Galfre et al., Methods Enzymol. 73:3-46, 1981).

The hybridoma obtained from cell fusion is selected by culturing thecells in a standard selection medium, for example, HAT culture medium(medium containing hypoxanthine, aminopterin, and thymidine). Theculture in this HAT medium is continued for a period sufficient enoughfor cells (non-fusion cells) other than the objective hybridoma toperish, usually from a few days to a few weeks. Then, the usual limitingdilution method is carried out, and the hybridoma producing theobjective antibody is screened and cloned.

Other than the above method for obtaining hybridomas, by immunizing ananimal other than humans with the antigen, a hybridoma producing theobjective human antibodies having the activity to bind to proteins canbe obtained by the method of sensitizing human lymphocytes, for example,human lymphocytes infected with the EB virus, with proteins,protein-expressing cells, or lysates thereof in vitro and fusing thesensitized lymphocytes with myeloma cells derived from human, forexample, U266, having a permanent cell division ability.

The monoclonal antibodies obtained by transplanting the obtainedhybridomas into the abdominal cavity of a mouse and extracting ascitescan be purified by, for example, ammonium sulfate precipitation, proteinA or protein G column, DEAE ion exchange chromatography, an affinitycolumn to which the protein of the present disclosure is coupled, and soon.

Monoclonal antibodies can be also obtained as recombinant antibodiesproduced by using the genetic engineering technique (see, for example,Borrebaeck C. A. K and Larrick, J. W., THERAPEUTIC MONOCLONALANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD(1990)). Recombinant antibodies are produced by cloning the encoding DNAfrom immunocytes, such as hybridoma or antibody-producing sensitizedlymphocytes, incorporating into a suitable vector, and introducing thisvector into a host to produce the antibody. The present disclosureencompasses such recombinant antibodies as well.

Antibodies or antibody fragments specific for a protein encoded by oneor more biomarkers can also be generated by in vitro methods such asphage display.

Moreover, the antibody of the present disclosure may be an antibodyfragment or modified-antibody, so long as it binds to a protein encodedby a biomarker of the invention. For instance, Fab, F(ab′)2, Fv, orsingle chain Fv (scFv) in which the H chain Fv and the L chain Fv aresuitably linked by a linker (Huston et al., Proc. Natl. Acad. Sci. USA,85:5879-5883, (1988)) can be given as antibody fragments. Specifically,antibody fragments are generated by treating antibodies with enzymes,for example, papain or pepsin. Alternatively, they may be generated byconstructing a gene encoding an antibody fragment, introducing this intoan expression vector, and expressing this vector in suitable host cells(see, for example, Co et al., J. Immunol., 152:2968-2976, 1994; Betteret al., Methods Enzymol., 178:476-496, 1989; Pluckthun et al., MethodsEnzymol., 178:497-515, 1989; Lamoyi, Methods Enzymol., 121:652-663,1986; Rousseaux et al., Methods Enzymol., 121:663-669, 1986; Bird etal., Trends Biotechnol., 9:132-137, 1991).

The antibodies may be conjugated to various molecules, such aspolyethylene glycol (PEG), fluorescent substances, radioactivesubstances, and luminescent substances. Methods to attach such moietiesto an antibody are already established and conventional in the field(see, e.g., U.S. Pat. Nos. 5,057,313 and 5,156,840).

Examples of methods that assay the antigen-binding activity of theantibodies include, for example, measurement of absorbance,enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA),radioimmunoassay (RIA), and/or immunofluorescence. For example, whenusing ELISA, a protein encoded by a biomarker of the invention is addedto a plate coated with the antibodies of the present disclosure, andthen, the antibody sample, for example, culture supernatants ofantibody-producing cells, or purified antibodies are added. Then,secondary antibody recognizing the primary antibody, which is labeled byalkaline phosphatase and such enzymes, is added, the plate is incubatedand washed, and the absorbance is measured to evaluate theantigen-binding activity after adding an enzyme substrate such asp-nitrophenyl phosphate. As the protein, a protein fragment, forexample, a fragment comprising a C-terminus, or a fragment comprising anN-terminus may be used. To evaluate the activity of the antibody of theinvention, BIAcore (Pharmacia) may be used.

By using these methods, the antibody of the invention and a samplepresumed to contain a protein of the invention are contacted, and theprotein encoded by a biomarker of the invention is detected or assayedby detecting or assaying the immune complex formed between theabove-mentioned antibody and the protein.

Mass spectrometry based quantitation assay methods, for example, but notlimited to, multiple reaction monitoring (MRM)-based approaches incombination with stable-isotope labeled internal standards, are analternative to immunoassays for quantitative measurement of proteins.These approaches do not require the use of antibodies and so theanalysis can be performed in a cost- and time-efficient manner (see, forexample, Addona et al., Nat. Biotechnol., 27:633-641, 2009; Kuzyk etal., Mol. Cell Proteomics, 8:1860-1877, 2009; Paulovich et al.,Proteomics Clin. Appl., 2:1386-1402, 2008). In addition, MRM offerssuperior multiplexing capabilities, allowing for the simultaneousquantification of numerous proteins in parallel. The basic theory ofthese methods has been well-established and widely utilized for drugmetabolism and pharmacokinetics analysis of small molecules.

Methods for detecting or measuring gene expression (e.g., mRNA orprotein expression) can optionally be performed in formats that allowfor rapid preparation, processing, and analysis of multiple samples.This can be, for example, in multi-welled assay plates (e.g., 96 wellsor 386 wells) or arrays (e.g., nucleic acid chips or protein chips).Stock solutions for various reagents can be provided manually orrobotically, and subsequent sample preparation (e.g., RT-PCR, labeling,or cell fixation), pipetting, diluting, mixing, distribution, washing,incubating (e.g., hybridization), sample readout, data collection(optical data) and/or analysis (computer aided image analysis) can bedone robotically using commercially available analysis software,robotics, and detection instrumentation capable of detecting the signalgenerated from the assay. Examples of such detectors include, but arenot limited to, spectrophotometers, luminometers, fluorimeters, anddevices that measure radioisotope decay. Exemplary high-throughputcell-based assays (e.g., detecting the presence or level of a targetprotein in a cell) can utilize ArrayScan® VTI HCS Reader or KineticScan®HCS Reader technology (Cellomics Inc., Pittsburgh, Pa.).

In some embodiments, the expression level of two genes, three genes,four genes, five genes, six genes, seven genes, eight genes, nine genes,10 genes, 11 genes, 12 genes, 13 genes, 14 genes, 15 genes, 16 genes, 17genes, 18 genes, 19 genes, 20 genes, 21 genes, 22 genes, 23 genes, atleast 24 genes, at least 25 genes or more, or at least two genes, atleast three genes, at least four genes, at least five genes, at leastsix genes, at least seven genes, at least eight genes, at least ninegenes, at least 10 genes, at least 11 genes, at least 12 genes, at least13 genes, at least 14 genes, at least 15 genes, at least 16 genes, atleast 17 genes, at least 18 genes, at least 19 genes, at least 20 genes,at least 21 genes, at least 22 genes, at least 23 genes, at least 24genes, or at least 25 genes or more can be assessed and/or measured.

To aid in detecting the presence or level of expression of one or moreof the genes depicted in Table 3, any part of the nucleic acid sequenceof the genes can be used, e.g., as hybridization polynucleotide probesor primers (e.g., for amplification or reverse transcription). Theprobes and primers can be oligonucleotides of sufficient length toprovide specific hybridization to an RNA, DNA, cDNA, or fragmentsthereof derived from a biological sample. Depending on the specificapplication, varying hybridization conditions can be employed to achievevarying degrees of selectivity of a probe or primer towards targetsequence. The primers and probes can be detectably-labeled with reagentsthat facilitate detection (e.g., fluorescent labels, chemical labels(see, e.g., U.S. Pat. Nos. 4,582,789 and 4,563,417), or modified bases).

Standard stringency conditions are described by Sambrook, et al. (supra)and Haymes, et al. Nucleic Acid Hybridization, A Practical Approach, IRLPress, Washington, D.C. (1985). In order for a nucleic acid molecule toserve as a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular hybridization conditions (e.g., solvent and saltconcentrations) employed.

Hybridization can be used to assess homology between two nucleic acidsequences. A nucleic acid sequence described herein, or a fragmentthereof, can be used as a hybridization probe according to standardhybridization techniques. The hybridization of a probe of interest(e.g., a probe containing a portion of a nucleotide sequence describedherein or its complement) to DNA, RNA, cDNA, or fragments thereof from atest source is an indication of the presence of DNA or RNA correspondingto the probe in the test source. Hybridization conditions are known tothose skilled in the art and can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Moderatehybridization conditions are defined as hybridization in 2× sodiumchloride/sodium citrate (SSC) at 30° C., followed by a wash in 1×SSC,0.1% SDS at 50° C. Highly stringent conditions are defined ashybridization in 6×SSC at 45° C., followed by a wash in 0.2×SSC, 0.1%SDS at 65° C.

Primers can be used in a variety of PCR-type methods. For example,polymerase chain reaction (PCR) techniques can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA The PCR primers are designed toflank the region that one is interested in amplifying. Primers can belocated near the 5′ end, the 3′ end or anywhere within the nucleotidesequence that is to be amplified. The amplicon length is dictated by theexperimental goals. For qPCR, the target length is closer to 100 bp andfor standard PCR, it is near 500 bp. Generally, sequence informationfrom the ends of the region of interest or beyond is employed to designoligonucleotide primers that are identical or similar in sequence toopposite strands of the template to be amplified. PCR primers can bechemically synthesized, either as a single nucleic acid molecule (e.g.,using automated DNA synthesis in the 3′ to 5′ direction usingphosphoramidite technology) or as a series of oligonucleotides. Forexample, one or more pairs of long oligonucleotides (e.g., >100nucleotides) can be synthesized that contain the desired sequence, witheach pair containing a short segment of complementarity (e.g., about 15nucleotides) such that a duplex is formed when the oligonucleotide pairis annealed. DNA polymerase is used to extend the oligonucleotides,resulting in a single, double-stranded nucleic acid molecule peroligonucleotide pair.

In addition, the nucleic acid sequences or fragments thereof (e.g.,oligonucleotide probes) can be used in nucleic acid arrays (such as thenucleic acid arrays described below under “Arrays”) for detection and/orquantitation of gene expression.

Cut-Off Values

As noted above, the methods described herein can involve, assessing theexpression level (e.g., mRNA or protein expression level) of one or moregenes (e.g., one or more genes depicted in Table 3), wherein theexpression level of one or more of the genes predicts the response of asubject to treatment comprising a lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate). “Assessing” caninclude, e.g., comparing the expression of one or more genes in a testbiological sample with a known or a control expression level (e.g., in areference biological sample) of the particular gene(s) of interest. Forexample, the expression level of one or more genes in a test biologicalsample can be compared to the corresponding expression levels in asubject who has responded or failed to respond to lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate), oran average or median expression level of multiple (e.g., two, three,four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 ormore) subjects, of the same species, who have responded or have failedto respond to lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate). Assessing can also include determining ifthe expression level of one or more genes (e.g., one or more genes asdepicted in Table 3) falls within a range of values predetermined aspredictive of responsiveness of a subject to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate). In some embodiments, assessing can be, or include,determining if the expression of one or more genes (e.g., one or more ofthe genes depicted in Table 3) falls above or below a predeterminedcut-off value. A cut-off value is typically an expression level of agene, or ratio of the expression level of a gene with the expressionlevel of another gene, above or below which is considered predictive ofresponsiveness of a subject to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).Thus, in accordance with the methods (and compositions) describedherein, a reference expression level of a gene (e.g., a gene depicted inTable 3) is identified as a cut-off value, above or below of which ispredictive of responsiveness to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).Some cut-off values are not absolute in that clinical correlations canstill remain significant over a range of values on either side of thecutoff however, it is possible to select an optimal cut-off value (e.g.varying H-scores) of expression levels of genes for a particular sampletypes. Cut-off values determined for use in the methods described hereincan be compared with, e.g., published ranges of expression levels butcan be individualized to the methodology used and patient population. Itis understood that improvements in optimal cut-off values could bedetermined depending on the sophistication of statistical methods usedand on the number and source of samples used to determine referencelevel values for the different genes and sample types. Therefore,established cut-off values can be adjusted up or down, on the basis ofperiodic re-evaluations or changes in methodology or populationdistribution.

The reference expression level of one or more genes can be determined bya variety of methods. The reference level can be determined bycomparison of the expression level of a gene of interest in, e.g.,populations of subjects (e.g., patients) that are responsive to atherapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate) or not responsive to a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof.This can be accomplished, for example, by histogram analysis, in whichan entire cohort of patients are graphically presented, wherein a firstaxis represents the expression level of a gene and a second axisrepresents the number of subjects in the cohort whose sample contain oneor more expression levels at a given amount. Determination of thereference expression level of a gene can then be made based on an amountwhich best distinguishes these separate groups. The reference level canbe a single number, equally applicable to every subject, or thereference level can vary, according to specific subpopulations ofsubjects. For example, older subjects can have a different referencelevel than younger subjects for the same metabolic disorder. Inaddition, a subject with more advanced disease (e.g., a more advancedform of a disease treatable by lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate)) can have adifferent reference value than one with a milder form of the disease.

Creating a Response Profile

The methods described herein can also be used to generate a lenvatinib(e.g., lenvatinib mesylate) therapy response profile for a subject. Theprofile can include information that indicates whether one or more ofthe mutations such as those listed in Tables 1 and 2 are present in asample from the subject; and/or information that indicates theexpression level of one or more genes (e.g., one or more genes depictedin Table 3); and/or the expression ratio of thyroglobulin in a sample(e.g., plasma, serum) of the subject post/pre-treatment with lenvatinibor a pharmaceutically acceptable salt thereof and/or the histologicalanalysis of any tumors (e.g., whether a thyroid cancer is a FTC or PTC).A lenvatinib therapy response profile can include the expression levelof one or more additional genes and/or other proteomic markers, serummarkers, or clinical markers. The response profiles described herein cancontain information on the expression or expression level of at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, or at least 25 genes listed in Table 3. Theresponse profiles described herein can also contain information on thepresence of mutations (if any) and the nature of the mutation(s) in anyone or more of the following genes: NRAS, KRAS, VHL, BRAF, ERBB2, PTENand MET. The resultant information (lenvatinib therapy response profile)can be used for predicting the response of a subject (e.g., a humanpatient) to a treatment comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate). In addition, theresponse profiles can be used in predicting the response of a subject toa variety of therapies and/or a variety of disease states since, e.g.,the expression levels of one or more of the genes (e.g., one or more ofthe genes depicted in Table 3), the mutations, the thyroglobulin levels,and/or the histological data examined can be indicative of suchresponses or disorders, whether or not physiologic or behavioralsymptoms of the disorder have become apparent.

It is understood that a lenvatinib (e.g., lenvatinib mesylate) responseprofile can be in electronic form (e.g., an electronic patient recordstored on a computer or other electronic (computer-readable) media suchas a DVD, CD, or floppy disk) or written form. The lenvatinib (e.g.,lenvatinib mesylate) response profile can also include information forseveral (e.g., two, three, four, five, 10, 20, 30, 50, or 100 or more)subjects (e.g., human patients). Such multi-subject response profilescan be used, e.g., in analyses (e.g., statistical analyses) ofparticular characteristics of subject cohorts.

Responsiveness of a subject to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) canbe classified in several ways and classification is dependent on thesubject's disease (e.g., thyroid cancer, a kidney cancer, or any otherof the diseases treatable by therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)),the severity of the disease, and the particular medicament the subjectis administered. In the simplest sense, responsiveness is any decreasein the disease state as compared to pre-treatment, andnon-responsiveness is the lack of any change in the disease state ascompared to pre-treatment. Responsiveness of a subject (e.g., a human)with a cancer can be classified based on one or more of a number ofobjective clinical indicia such as, but not limited to, tumor size,Clinical Benefit (CB), Overall Survival (OS), Progression Free Survival(PFS), Disease Control Rate (DCR), Time-To-Response (TTR), TumorShrinkage (TS), or Tumor Response (TR).

“Clinical benefit” refers to having one of the followingstatuses—Complete Response (CR), Partial Response (PR); or StableDisease (SD) with 6 months or more progression free survival (PFS).“Complete Response” means complete disappearance of all target lesions.“Partial Response” means at least 30% decrease in the sum of the longestdiameter (LD) of target lesions, taking as reference the baseline summedLD. “Progressive Disease” (PD) means at least 20% increase in the sum ofthe LD of target lesions, taking as reference the smallest summed LDrecorded since the treatment started, or the appearance of one or morenew lesions. “Stable Disease” means neither sufficient shrinkage of thetarget lesions to qualify for PR nor sufficient increase to qualify forprogressive disease (PD), taking as reference the smallest summed LDsince the treatment started.

“Overall Survival” (OS) is defined as the time from randomization untildeath from any cause. “Randomization” means randomization of a patientinto a test group or a control group when therapy plan for a patient isdetermined.

“Progression Free Survival” (PFS) refers to the period from start dateof treatment to the last date before entering PD status.

“Disease Control Rate” (DCR) is defined as CR or PR or SD for 7 weeks.

“Time-To-Response” (TTR) is defined as the time from the date ofinitiation of treatment to the date when criteria for response (CR orPR) are first met.

“Tumor shrinkage” (TS) means percent change of sum of diameters oftarget lesions, taking as reference the baseline sum diameters.

“Tumor response” (TR) compares subjects with “Partial Response” (PR)with subjects with either Stable Disease (SD) or Progressive Disease(PD).

Methods of Treatment

The methods disclosed herein enable the assessment of a subject forresponsiveness to lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate). A subject who is likely to respondto lenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) can be administered lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).

The methods of this disclosure also enable the classification ofsubjects into groups of subjects that are more likely to benefit, andgroups of subjects that are less likely to benefit, from treatment withlenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate). The ability to select such subjects from a pool ofsubjects who are being considered for treatment with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) isbeneficial for effective treatment.

The methods of this disclosure can also be used to determine whether tocontinue treatment with lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate) after administering this therapy fora short period of time and determining based on the expression profileof one or more of the biomarkers described above post-treatment orpost-treatment versus pre-treatment whether this therapy is more likelyor less likely to benefit the patient.

Lenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) shows potent anti-tumor effects in xenograft modelsof various tumors by inhibiting angiogenesis. The subjects who areconsidered for treatment with lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate) include, but are notlimited to, subjects having, suspected of having, or likely to develop athyroid cancer or a kidney cancer (e.g., renal cell carcinoma).

In one embodiment, the subject to be treated with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)has, is suspected of having, or is likely to develop a thyroid cancer.Thyroid cancer is a cancerous tumor or growth located within the thyroidgland. It is the most common endocrine cancer and is one of the fewcancers that has increased in incidence rates over recent years. Itoccurs in all age groups from children through seniors. The AmericanCancer Society estimates that there were about 44,670 new cases ofthyroid cancer in the U.S. in 2010. Of these new cases, about 33,930were in women and about 10,740 in men. About 1,690 people (960 women and730 men) died of thyroid cancer in 2010. Many patients, especially inthe early stages of thyroid cancer, do not experience symptoms. However,as the cancer develops, symptoms can include a lump or nodule in thefront of the neck, hoarseness or difficulty speaking, swollen lymphnodes, difficulty swallowing or breathing, and pain in the throat orneck. There are several types of thyroid cancer: papillary, follicular,medullary, anaplastic, and variants. Papillary carcinoma is the mostcommon type accounting for approximately 85% of all thyroid cancers, andusually affects women of childbearing age. It spreads slowly and is theleast dangerous type of thyroid cancer. Follicular carcinoma accountsfor about 10% of all cases and is more likely to come back and spread.Medullary carcinoma is a cancer of nonthyroid cells that are normallypresent in the thyroid gland. This form of the thyroid cancer tends tooccur in families. It requires different treatment than other types ofthyroid cancer. Anaplastic carcinoma (also called giant and spindle cellcancer) is the most dangerous form of thyroid cancer. It is rare, anddoes not respond to radioiodine therapy. Anaplastic carcinoma spreadsquickly and invades nearby structures such as the windpipe (trachea),causing breathing difficulties. Variants include tall cell, insular,columnar, and Hurthle cell. Lenvatinib or a pharmaceutically acceptablesalt thereof (e.g., lenvatinib mesylate) can be used to treat a subjecthaving, suspected of having, or likely to develop any of theabove-described thyroid cancers. In certain embodiments, the subject tobe treated with lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate) has, is suspected of having, or is likely todevelop a differentiated thyroid cancer. In other embodiments, thesubject to be treated with lenvatinib or a pharmaceutically acceptablesalt thereof (e.g., lenvatinib mesylate) has, is suspected of having, oris likely to develop a medullary thyroid cancer. In one embodiment, thesubject to be treated with lenvatinib or a pharmaceutically acceptablesalt thereof (e.g., lenvatinib mesylate) has, is suspected of having, oris likely to develop a papillary thyroid cancer. In another embodiment,the subject to be treated with lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate) has, is suspected ofhaving, or is likely to develop a follicular thyroid cancer. In anotherembodiment, the subject to be treated with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)has, is suspected of having, or is likely to develop a Hürthle-cellthyroid cancer.

In one embodiment, the subject to be treated with lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate)has, is suspected of having, or is likely to develop a kidney cancer.Kidney cancer is usually defined as a cancer that originates in thekidney. The two most common types of kidney cancer, reflecting theirlocation within the kidney, are renal cell carcinoma (RCC, also known ashypernephroma) and urothelial cell carcinoma (UCC) of the renal pelvis.Other, less common types of kidney cancer include: Squamous cellcarcinoma, Juxtaglomerular cell tumor (reninoma), Angiomyolipoma, Renaloncocytoma, Bellini duct carcinoma, Clear cell sarcoma of the kidney,Mesoblastic nephroma, Wilms' tumor, and mixed epithelial stromal tumor.RCC is a kidney cancer that originates in the lining of the proximalconvoluted tubule, the very small tubes in the kidney that filter theblood and remove waste products. RCC is the most common type of kidneycancer in adults, responsible for approximately 80% of cases. It is alsoknown to be the most lethal of all the genitourinary tumors. Initialtreatment is most commonly a radical or partial nephrectomy and remainsthe mainstay of curative treatment. Where the tumor is confined to therenal parenchyma, the 5-year survival rate is 60-70%, but this islowered considerably where metastases have spread. It is resistant toradiation therapy and chemotherapy, although some cases respond toimmunotherapy. Lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate) can be used to treat a subject having,suspected of having, or likely to develop any of the above-describedkidney cancers. In a specific embodiment, the subject to be treated withlenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) has, is suspected of having, or is likely todevelop a renal cell carcinoma.

If the subject is more likely to respond to a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (based onpresence of mutational biomarkers and/or expression levels/ratios of thebiomarkers described above), the subject can then be administered aneffective amount of the lenvatinib compound (e.g., lenvatinib mesylate).An effective amount of the compound can suitably be determined by ahealth care practitioner taking into account, for example, thecharacteristics of the patient (age, sex, weight, race, etc.), theprogression of the disease, and prior exposure to the drug. If thesubject is less likely to respond to a therapy comprising lenvatinib ora pharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate),the subject can then be optionally administered a therapy that does notcomprise lenvatinib. These therapies include, but are not limited to,radioactive iodine, doxorubicin, carboplatin, cisplatin, paclitaxel,sorafenib, docetaxel, trastumab, interleukin-2, interferon, everolimus,sunitinib, pazopanib, vandetanib, and “standard of care” treatment(i.e., prevailing standard of care as determined by the health carepractitioner or as specified in the clinical study) such asinvestigational drugs and chemotherapy.

Subjects of all ages can be affected by disorders treatable bylenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate). Therefore, a biological sample used in a methodsdescribed herein can be obtained from a subject (e.g., a human) of anyage, including a child, an adolescent, or an adult, such as an adulthaving, or suspected of having, a disease (e.g., papillary thyroidcancer, renal cell carcinoma) treatable by lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).

The methods can also be applied to individuals at risk of developing acancer treatable by lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate). Such individuals include those whohave (i) a family history of (a genetic predisposition for) suchdisorders or (ii) one or more risk factors for developing suchdisorders.

After classifying or selecting a subject based on whether the subjectwill be more likely or less likely to respond to lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate), amedical practitioner (e.g., a doctor) can administer the appropriatetherapeutic modality to the subject. Methods of administering lenvatinibtherapies are well known in the art.

It is understood that any therapy described herein (e.g., a therapycomprising a lenvatinib or a therapy that does not comprise alenvatinib) can include one or more additional therapeutic agents. Thatis, any therapy described herein can be co-administered (administered incombination) with one or more additional therapeutic agents such as, butnot limited to, doxorubicin, carboplatin, cisplatin, paclitaxel,docetaxel, trastumab, interleukin-2, interferon and everolimus.Furthermore, any therapy described herein can include one or more agentsfor treating, for example, pain, nausea, and/or one or more side-effectsof a therapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate).

Combination therapies (e.g., co-administration of a therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) and one or more additional therapeutic agents) canbe, e.g., simultaneous or successive. For example, lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) andone or more additional therapeutic agents can be administered at thesame time or a lenvatinib compound (e.g., lenvatinib mesylate) can beadministered first in time and the one or more additional therapeuticagents administered second in time. In some embodiments, the one or moreadditional therapeutic agents can be administered first in time andlenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) administered second in time.

In cases where the subject predicted to respond to a lenvatinib (e.g.,lenvatinib mesylate) therapy has been previously administered one ormore non-lenvatinib therapies, the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) canreplace or augment a previously or currently administered therapy. Forexample, upon treating with the therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate),administration of the one non-lenvatinib therapies can cease ordiminish, e.g., be administered at lower levels. Administration of theprevious therapy can be maintained while the therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) is administered. In some embodiments, a previoustherapy can be maintained until the level of the therapy comprisinglenvatinib or a pharmaceutically acceptable salt thereof (e.g.,lenvatinib mesylate) reaches a level sufficient to provide a therapeuticeffect.

Arrays

Nucleic acid arrays including the nucleic acid biomarkers disclosedherein are useful in, e.g., detecting gene expression and/or measuringgene expression levels. The arrays are also useful for e.g., inpredicting the response of a subject to a therapy comprising lenvatinibor a pharmaceutically acceptable salt thereof (e.g., lenvatinibmesylate), for identifying subjects who can benefit from a therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof(e.g., lenvatinib mesylate), and for steering subjects who would notlikely benefit from a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate) toother cancer therapies.

An array is an orderly arrangement of samples where matching of knownand unknown DNA samples is done based on base pairing rules (e.g.,Adenosine pairs with Thymine or Uracil; Guanosine pairs with Cytosine).A typical microarray experiment involves the hybridization of an mRNA, acDNA molecule, or fragments thereof, to a DNA template from which it isoriginated or derived. Many DNA samples are used to construct an array.An array experiment makes use of common assay systems such asmicroplates or standard blotting membranes. The sample spot sizes aretypically less than 200 microns in diameter and the array usuallycontains thousands of spots. Thousands of spotted samples known asprobes (with known identity) are immobilized on a substrate (e.g., amicroscope glass slides, silicon chips, nylon membrane). The spots canbe DNA, cDNA, or oligonucleotides. These are used to determinecomplementary binding of the unknown sequences thus allowing parallelanalysis for gene expression and gene discovery. An experiment with asingle DNA chip can provide information on thousands of genessimultaneously. An orderly arrangement of the probes on the support isimportant as the location of each spot on the array is used for theidentification of a gene. The amount of mRNA bound to each site on thearray indicates the expression level of the various genes that areincluded on the array. By using an array containing many DNA samples,one can determine, in a single experiment, the expression levels ofhundreds or thousands of genes by measuring the amount of mRNA bound toeach site on the array. With the aid of a computer, the amount of mRNAbound to the spots on the microarray can be precisely measured,generating a profile of gene expression in the cell.

The two main DNA microarray platforms that are generally used are cDNAand oligonucleotide microarrays. cDNA microarrays are made with longdouble-stranded DNA molecules generated by enzymatic reactions such asPCR (Schena, M. et al., Science, 270:467-470 (1995)), whileoligonucleotide microarrays employ oligonucleotide probes spotted byeither robotic deposition or in situ synthesis on a substrate (Lockhart,D. J. et al., Nat. Biotechnol., 14, 1675-1680 (1996)).

Kits

This application also provides kits. In some embodiments, the kitsinclude probes that can be used to identify or detect any of thebiomarkers of Table 3. In some embodiments, the kits include primersthat can be used to amplify the region containing any of the mutationslisted in Table 1 and/or Table 2. In some embodiments, the kits includeany of the nucleic acid arrays described herein. In certain embodiments,the kits include antibodies that can be used to detect thyroglobulin orto detect any of the biomarkers of Table 3 or their expression orexpression levels. In some embodiments, the kits include probes andantibodies that can be used to identify or detect any of the biomarkersof Table 3 or their expression or expression levels. The kits can,optionally, contain instructions for detecting and/or measuring thelevel of one or more genes in a biological sample.

The kits can optionally include, e.g., a control biological sample orcontrol labeled-amplicon set containing known amounts of one or moreamplicons recognized by nucleic acid probes of the array. In someinstances, the control can be an insert (e.g., a paper insert orelectronic medium such as a CD, DVD, or floppy disk) containingexpression level ranges of one or more genes predictive of a response toa therapy comprising lenvatinib or a pharmaceutically acceptable saltthereof (e.g., lenvatinib mesylate).

In some embodiments, the kits can include one or more reagents forprocessing a biological sample. For example, a kit can include reagentsfor isolating a protein from a biological sample and/or reagents fordetecting the presence and/or amount of a protein in a biological sample(e.g., an antibody that binds to the protein that is the subject of thedetection assay and/or an antibody that binds the antibody that binds tothe protein).

In some embodiments, the kits can include a software package foranalyzing the results of, e.g., a microarray analysis or expressionprofile.

The kits can also include one or more antibodies for detecting theprotein expression of any of the genes described herein. For example, akit can include (or in some cases consist of) a plurality of antibodiescapable of specifically binding to one or more proteins encoded by anyof the genes depicted in Table 3 and optionally, instructions fordetecting the one or more proteins and/or a detection antibodycomprising a detectably-labeled antibody that is capable of binding toat least one antibody of the plurality. In some embodiments, the kitscan include antibodies that recognize one, at least two, at least three,at least four, at least five, at least six, at least seven, at leasteight, at least nine, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45 or 46 proteins encoded by genes depictedin Table 3.

The kits described herein can also, optionally, include instructions foradministering a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof (e.g., lenvatinib mesylate), where theexpression level of one or more genes detectable by the array predictsthat a subject will respond to a therapy comprising lenvatinib or apharmaceutically acceptable salt thereof (e.g., lenvatinib mesylate).

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1: Mutation Status as Predictive Biomarkers forResponsiveness to Therapy Comprising E7080

Purpose:

Tumor response and prolonged disease stabilization were observed indifferentiated thyroid cancer patients treated in phase II with E7080(lenvatinib). This experiment was directed at identifying amino acidmutations that are useful in predicting whether subjects respond totreatment with E7080 using three criteria of response: best overallresponse, tumor shrinkage, and progression free survival.

Materials and Methods:

Tissue samples were obtained at surgery before the patients had receivedany therapy comprising E7080 and were routinely processed with formalinfixed, paraffin embedded tissues (FFPE). The protocol that was used wasapproved by the institutional review board, and informed consent wasobtained from each subject. Tumor tissue samples from 27 patients, forwhich tissues were available, were used for mutation analysis. DNA wasisolated from FFPE tumor blocks collected from patients participating inthe trial. Genomic DNA was extracted from two to five 10 micronunstained sections by deparaffinization and Qiagen DNA Mini Kit TissueProtocol with minor modification. For mutation detection, the SEQUENOM®(San Diego, Calif.) platform and the OncoCarta™ Panel v1.0 andOncoCarta™ Panel v3.0 were used (see,www.sequenom.com/Files/Genetic-Analysis—Graphics/All-Application—PDFs/AssayExplorer2010_1110-Web/.Sequenom's OncoCarta™ Panels are a set of pre-designed and pre-validatedassays for efficient mutation screening. The OncoCarta™ Panel v1.0 genesand the number of mutations (in parentheses) are: ABL1 (14); AKT1 (7);AKT2 (2); BRAF (25); CDK4 (2); EGFR (40); ERBB2 (9); FGFR1 (2); FGRF3(7); FLT3 (3); HRAS (10); JAK2 (1); KIT (32); KRAS (16); MET (5); NRAS(19); PDGFRA (11); PIK3CA (14); and RET (6). The OncoCarta™ Panel v3.0genes and the number of mutations (in parentheses) are: ABL1 (2) AKT1(1); APC (12); BRAF (19); CDKN2A(7); CSFIR (6); CTTNB1 (28); EGFR (32);ERBB2 (2); FLT3 (3); HRAS (2); JAK3 (3); KIT (3); KRAS (5); MET (6);MLH1 (1); MYC (6); PDGFRA (11); PIK3CA (4); PTEN (14); RB1 (11); RET(13); SRC (1); STK11 (12); P53 (7); and VH1 (7). OncoCarta™ Panel v3.0genes and the number of mutations (in parentheses) are: ABL1 (2) AKT1(1); APC (12); BRAF (19); CDKN2A(7); CSF1R (6); CTTNB1 (28); EGFR (32);ERBB2 (2); FLT3 (3); HRAS (2); JAK3 (3); KIT (3); KRAS (5); MET (6);MLH1 (1); MYC (6); PDGFRA(11); PIK3CA (4); PTEN (14); RB1 (11); RET(13); SRC (1); STK11 (12); P53 (7); and VH1 (7).

DNA was amplified using the OncoCarta™ PCR primer pools, unincorporatednucleotides were inactivated by shrimp alkaline phosphatase (SAP), and asingle base extension reaction was performed using extension primersthat hybridize immediately adjacent to the mutations and a custommixture of nucleotides. Salts were removed by the addition of a cationexchange resin. Multiplexed reactions were spotted onto theSpectroChipII, and mutations, if present, were resolved by MALDI-TOF onthe Compact Mass Spectrometer (Sequenom®, San Diego, Calif.). TheOncoCarta™ Panel v1.0 (Sequenom®, San Diego, Calif.) consists of 24pools of primer pairs and 24 pools of extension primers, and has thecapacity to detect 225 mutations in 19 genes. The OncoCarta™ Panel v 3.0consists of 24 pools of primer pairs and 24 pools of extension primers,and has the capacity to detect 218 mutations in 26 genes. Each poolconsists of 5-9 primer pairs in the PCR reaction. Two types of assayshave been designed in the OncoCarta panel, referred to as simple andcomplex. The simple assays are those in which a single assay is able todetect the amino acid changes at that codon. The complex assays arethose that require more than one assay to identify codon changes ordeletions and insertion, and thus are able to detect multiple differentamino acid substitutions or deletions. An example of a complex assayinvolves the use of the KRAS_1 and KRAS_2 assays, which interrogates 2different nucleotide positions within codon 12 and together identify allcodon 12 amino acid changes. In the KRAS G12R mutation, the mutantallele has the codon CGT (Arginine) in contrast to the wild type allelewhich has the GGT (Glycine) codon. So, the first nucleotide of codon 12needs to be “C” and second nucleotide needs to be “G”. The KRAS_2 assayreveals which nucleotide is incorporated into the first nucleotide ofthe codon 12, and the KRAS_1 assay is for the second nucleotide.Together, the data from these two assays detects the G12R substitution.For the BRAF V600E mutation (GTG to GAG), BRAF_16 assay (for the firstnucleotide of the codon 600) identifies “G” and BRAF_15 (for the secondnucleotide) identifies “A”. For the VHL P81S mutation, the mutant allelehas TCG (Serine) whereas the wild type allele has CCG (Proline). TheVHL_498 assay reveals which nucleotide is incorporated into the firstnucleotide of the codon 81. For the KRAS Q61R mutation, the KRAS_7 assaydiscriminates the mutant allele (CGA, Arginine) from the wild typeallele (CAA, Glutamine) by examining nucleotide variation in the secondposition of the codon. In the case of NRAS codon 61, there are threeknown mutations, Q61L (CAA to CTA), Q61R (CAA to CGA) and Q61P (CAA toCCA). The NRAS_6 assay detects nucleotide variation in the secondposition of the codon. Even more complex assays are also included inOncoCarta™, which interrogate insertions and deletions within the EGFRgene.

Data analysis was performed using MassArray Type Analyzer software(Sequenom®), which facilitates visualization of data patterns as well asthe raw spectra. All mutations from the Onco mutation report werereviewed manually to identify “real” mutant peaks from salt peaks orother background peaks.

The period during which a patient takes E7080 was artificially dividedinto different Cycles for ease of evaluation and tracking. Patientsreceived E7080 at a dose of 24 mg oral once daily in 28 day cycles. Foranalysis purposes, assessments of clinical outcomes were performed atthe time of the 9 month and at 14 month minimum follow-up. For the E7080Thyroid Cancer trial, each Cycle is 28 days (4 weeks) so Day 1-28 iscycle 1; Day 29 is Day 1 of Cycle 2; and Day 57 is Day 1 of Cycle 3.Blood samples were collected for pharmacokinetic (PK) analysis on Cycle1 Days 1 and 8, Cycle 2 Day 1, and Cycle 3 Day 1. A total of 9 samplesper patient were collected as follows: Cycle 1 Day 1: immediately priorto the dose of E7080, and at 0.5 and 2 hours following the first dose ofE7080 (post-dose); Cycle 1 Day 8: immediately prior to the dose ofE7080; Cycle 2 Day 1: immediately prior to the dose of E7080, 0.5 and 2hours post-dose; Cycle 3 Day 1: immediately prior to the dose of E7080and 2 hours post-dose. For analysis of progression free survival, PKparameter was used as a covariate in Cox proportional hazards model.

The three criteria of response: best overall response, tumor shrinkage,and progression free survival are defined below.

“Best Overall Response” (BOR) refers to having one of the followingstatuses—Complete Response (CR), Partial Response (PR), Stable Disease(SD) or Progressive Disease (PD).

“Clinical benefit” (CB) refers to having one of the followingstatuses—Complete Response (CR), Partial Response (PR); or StableDisease (SD) with 6 months or more progression free survival (PFS).

“Complete Response” means complete disappearance of all target lesions.

“Partial Response” means at least 30% decrease in the sum of the longestdiameter (LD) of target lesions, taking as reference the baseline summedLD.

“Progressive Disease” (PD) means at least 20% increase in the sum of theLD of target lesions, taking as reference the smallest summed LDrecorded since the treatment started, or the appearance of one or morenew lesions.

“Stable Disease” means neither sufficient shrinkage of the targetlesions to qualify for PR nor sufficient increase to qualify forprogressive disease (PD), taking as reference the smallest summed LDsince the treatment started.

“Progression Free Survival” (PFS) refers to the period from start dateof treatment to the last date before entering PD status.

“Tumor shrinkage” (TS) means percent change of sum of diameters oftarget lesions, taking as reference the baseline sum diameters.

Results:

A total of 443 mutations among 33 genes were tested using OncoCarta™Panel v1.0 and OncoCarta™ Panel v3.0 mutation panel and mutations werefound in 16 of the 23 subjects we examined. From 27 patients, 23 patientsamples were analyzed using OncoCarta™ Panel v1.0 and using OncoCarta™Panel v3.0. 12 mutations among 10 genes were identified and are listedin Table 4. 7 patients were wild type for all genes tested.

TABLE 4 Summary of Mutations Found in DTC Patients No. of pa- Mutationtient BRAF NRAS KRAS VHL Other mutation 1 WT Q61P WT P81SERBB2(S779_P780insVGS) 2 WT Q61R WT P81S PTEN(N323fs*2) 3 WT Q61R WT WT4 WT Q61R WT WT 5 WT Q61R WT WT 6 WT Q61R WT P81S 7 V600E WT WT WT 8V600E WT WT WT myc(A59V) 9 V600E WT WT WT 10 V600E WT WT WT 11 WT WT WTP81S 12 WT WT 061R WT 13 WT WT G12R WT 14 WT WT WT WTPI3KCA(E545A.E545G) 15 WT WT WT WT TP53(R248W) 16 WT WT WT WTMET(T1010I.T992I)

The Genbank® accession number for: BRAF is NM_004333.3; for NRAS isNM_002524.2; for KRAS is NM_033360.2 (variant A) and NM_004985.3(variant B); and for VHL is NM_000551.2 (variant 1) and NM_198156.1(variant 2).

Best overall response was significantly better in patients whose tumorhad mutations in any of KRAS, NRAS, BRAF, VHL-1 genes (Fishers exacttest) as shown in Table 5. For example, 8 out of 8 patients with eitheran NRAS or KRAS mutation had partial response (PR), while only 5 out of14 patients had PR without mutations in NRAS and KRAS.

TABLE 5 Mutation and Best Overall Response (BOR) Mutation BOR p-value(any of gene) (fisher's exact) BRAF.NRAS 0.038 KRAS.NRAS 0.007BRAF.KRAS.NRAS 0.010 BRAF.VHL 0.042 BRAF.KRAS.VHL 0.034 KRAS.NRAS.VHL0.020 BRAF.KRAS.NRAS.VHL 0.012 Mutation (any of gene) BOR WT MUBRAF.NRAS PR 5 8 SD 6 1 PD 2 0 KRAS.NRAS PR 5 8 SD 7 0 PD 2 0BRAF.KRAS.NRAS PR 3 10 SD 6 1 PD 2 0 BRAF.VHL PR 10 6 SD 5 0 PD 0 2BRAF.KRAS.VHL PR 8 8 SD 5 0 PD 0 2 KRAS.NRAS.VHL PR 5 8 SD 7 0 PD 1 1BRAF.KRAS.NRAS.VHL PR 3 10 SD 6 1 PD 1 1 * Tumor size of 1 wild typepatient was not evaluable

Patients with mutations in either NRAS, either NRAS or KRAS, or eitherBRAF, KRAS, or NRAS, had a larger decrease of tumor size indicated aspercent change in tumor shrinkage, compared with patients that were wildtype for NRAS, KRAS and BRAF (see, Table 6).

TABLE 6 Mutation and % of Maximum Tumor Shrinkage % of tumor shrinkageGene Wild (Median) p (any of gene) type* Mutation wild type mutationvalue NRAS 16 6 −35.5 −45.5 0.042 KRAS.NRAS 14 8 −31.0 −44.5 0.022*Tumor size of 1 wild type patient was not evaluable

The association of gene mutations with progression free survival oftreated patients was analyzed first by performing Logrank test and thenusing Cox proportional hazards model with or without covariates. Thecovariate used are the following PK parameters: cycle 1 day 1 Cmax(E7080 concentration 2 hr after dosing on cycle 1 day 1: MAX1); cycle1day1 Ctrough (E7080 concentration before dosing on cycle1 day 8:MIN1);cycle2 day1 Cmax (E7080 concentration 2 hrs after dosing on cycle2 day1:MAX2); cycle2 day1 Ctrough (E7080 concentration before dosing on cycle2 day 1:MIN2). The Logrank test demonstrated that patients withmutations in NRAS alone or with mutations in either NRAS or KRAS hadbetter progression free survival than those patients who were wild typefor NRAS or KRAS (FIG. 1 ). Cox proportional hazards model alsodemonstrated that patients with mutations in either NRAS, KRAS, or VHLand mutations in either NRAS, KRAS, or BRAF had better progression freesurvival than those who did not have mutations based on cox proportionhazard test with a covariate of cycle1 day1 Ctrough (MIN1), since wildtype set as 1 and mutation set as 0 and coefficients lower than 0 makeslower hazard for mutation (see, Table 7).

TABLE 7 Gene Mutation and Progression Free Survival Mutation PK (any ofgene) n Coef p value MIN1 KRAS.NRAS 23 −1.586 0.043 NRAS 23 −2.091 0.047BRAF.KRAS.NRAS 23 −1.428 0.032 0.037 KRAS.NRAS 23 −2.629 0.021 0.007KRAS.NRAS.VHL 23 −1.700 0.043 0.018Conclusion:

Mutations in a small set of genes, such as anyone or a combination ofNRAS, KRAS, BRAF, and VHL in thyroid tumors are useful in predicting theclinical response of a patient presenting with, suspected of having, orat risk of developing, thyroid cancer to a therapy comprising E7080.

Example 2: Thyroglobulin as a Biomarker for Therapy Comprising E7080 inThyroid Tumors

Purpose:

This experiment was directed at determining whether changes inthyroglobulin levels are predictive of whether patients respond totreatment with E7080 using three criteria of clinical response: tumorresponse, tumor shrinkage, and progression free survival.

Material and Methods:

The serum for the thyroglobulin assays was collected within 72 hoursprior to Day 1 of all cycles. 24 mg of E7080 was administered orally,daily continuously in 28-day cycles. Serum samples from 58 patients wereused for thyroglobulin measurements. 6 mL of blood was drawn into redtop tubes and let to clot at room temperature for at least 30 min.Within 60 minutes of sample collection, the tubes were centrifuged for15 minutes at 20-25° C. at 1000×g. The supernatant was drawn withoutdisturbing the pellet, put into sample tubes with serum filter andshipped frozen to the central lab for testing on the day of collection.The level of thyroglobulin was detected by a solid-phase,chemiluminescent immunometric assay (IMMULITE 2000 Thyroglobulin AssaySystem, Siemens) following standard operating procedure. In brief, serumthyroglobulin was captured by beads coated with anti-thyroglobulinantibody and detected using anti-thyroglobulin antibody linked toalkaline phosphatase. The level of serum thyroglobulin was calculatedfrom a standard curve generated with lyophilized thyroglobulin. Serumthyroglobulin was stable for up to 2 months when stored frozen for theassay. The sensitivity of this assay was 0.2 ng/ml.

Results:

From 58 patient samples, 50 pre- and post-treatment blood samples atmaximum were used for these analyses. Dramatic changes in thyroglobulinlevels were observed 29 days after the start of treatment with E7080.Thyroglobulin levels significantly decreased 48 days (within 2 cycles)after treatment with E7080 and the decrease continued to cycle 8.

Changes in thyroglobulin levels in groups of patient, who have partialresponse (PR) with E7080, was significantly lower than those in groupsof patients, who have either stable disease (SD) or progressive disease(PD) (Mann Whitney U test, FIG. 2 ; ∘ median of PR groups, ● median ofOthers—SD or PD and Table 8, top). Also changes in thyroglobulin levelswere associated with tumor shrinkage after completion of cycle 2 (56days after treatment) (Speaman's rank correlation test, Table 8,middle). Cox proportional hazard test demonstrated that progression freesurvival of patients was associated with changes in thyroglobulin levels28 days after treatment with E7080, indicating larger decrease predictlonger PFS (Table 8, bottom).

TABLE 8 Changes in Thyroglobulin Levels After E7080 Treatment (a) Largedecrease of thyroglobulin level in group of patients with PR PR groupOther group Factor Time point n median IQR Median IQR p valueThyroglobulin CYCLE2 DAY 1/CYCLE1 DAY 1 49 0.195 0.233 0.288 0.460 0.154CYCLE3 DAY 1/CYCLE1 DAY 1 44 0.109 0.144 0.221 0.243 0.034 CYCLE4 DAY1/CYCLE1 DAY 1 44 0.079 0.112 0.227 0.390 0.007 CYCLE5 DAY 1/CYCLE1 DAY1 42 0.106 0.119 0.332 0.403 0.019 CYCLE6 DAY 1/CYCLE1 DAY 1 40 0.0780.133 0.198 0.235 0.044 (b) Association of large decrease ofthyroglobulin level to % of tumor shrinkage maximum tumor shrinkageFactor Time point Correlation (r) p value Thyroglobulin CYCLE2 DAY1/CYCLE1 DAY 1 0.163 0.280 CYCLE3 DAY 1/CYCLE1 DAY 1 0.342 0.026 CYCLE4DAY 1/CYCLE1 DAY 1 0.467 0.002 CYCLE5 DAY 1/CYCLE1 DAY 1 0.443 0.004CYCLE6 DAY 1/CYCLE1 DAY 1 0.358 0.023 (c) Association of a change ofthyroglobulin level to PFS after 1 cycle treatment with E7080 FactorTime & category n Coef p value Thyroglobulin CYCLE2 DAY 1/CYCLE1 DAY 149 0.995 0.039Conclusion:

Changes in thyroglobulin levels following E7080 therapy are associatedto tumor response, decrease of tumor size, and progression free survivaland may be used to predict clinical response as early as 4 weeks aftertreatment with E7080. Thus, thyroglobulin levels are helpful inassessing whether to continue therapy comprising E7080.

Example 3: Cytokine, Chemokine, and Angiogenic Factors (BloodBiomarkers) as Predictive and Response Biomarkers of E7080

Purpose:

Angiogenesis is regulated by signaling through multiple growth factorreceptors, such as VEGF and FGF receptor. VEGF receptor signaling isalso associated with immune cell function. The purpose of this analysiswas to measure cytokine, chemokine and angiogenic factors (collectivelyreferred to herein as “blood biomarkers”) in serum samples obtained frompatients in clinical trials at both pre- and post-treatment with E7080and to identify blood biomarkers which can be used to predict whetherpatients will respond to treatment with E7080. For these analyses, threecriteria of response were employed, namely: tumor response, % of tumorshrinkage and progression free survival.

“Tumor response” (TR) compares subjects with “Partial Response” (PR)with subjects with either Stable Disease (SD) or Progressive Disease(PD).

Materials and Methods:

Patients received E7080 at a dose of 24 mg oral once daily in 28 daycycles. Serum samples were collected at Cycle 1 Day 1 (pre-treatment),Cycle 1 Day 8 and Cycle 2 Day8 (i.e., day 36 post-treatment). 7.5 mL ofblood was drawn into serum stainless steel tube (SST) and let to clot atroom temperature for at least 30 min. Within 60 minutes of samplecollection, the tubes were centrifuged for 10 minutes at 20-25° C. at1400×g. The supernatant was drawn without disturbing the pellet and theserum was mixed and divided into two sample tubes and stored frozen(−70° C.) at an upright position until further treatment for analysis.Serum samples from 58 patients were used for blood biomarker analysis.Serum from Cycle 1 day 1 (baseline), cycle 1 day 8, and cycle 2 day 8were used in this analysis. The association of progression free survivalwith or without a covariate was analyzed. The covariates used are thefollowing PK parameters: cycle 1 day 1 Cmax (E7080 concentration 2 hrafter dosing on cycle 1 day 1: MAX1); cycle1 day 1 Ctrough (E7080concentration before dosing on cycle1 day 8:MIN1); cycle2 day 1 Cmax(E7080 concentration 2 hrs after dosing on cycle2 day 1:MAX2); cycle2day 1 Ctrough (E7080 concentration before dosing on cycle 2 day 1:MIN2).Serum samples were tested in batch format where all timepoints from thesame subject were assayed on the same day. On the day of assay, sampleswere removed from −80° C. and allowed to thaw and reach roomtemperature. The serum samples were tested using the followingcommercial assay kits as per the manufacturer's instructions: HumanSoluble Tie-2 ELISA (R&D Systems Cat. No. DTE200), Human Angiopoietin-1ELISA (R&D Systems Cat. No. DANG10), Human FGF23 ELISA, Human SDF-1aELISA Human Angiopoietin-2 ELISA (R&D Systems Cat. No. DANG20), HumanSoluble Receptor Multiplex (Millipore Cat. No. HSCR-32K; sVEGF R1, sVEGFR2 and sVEGF R3 only), Human Cytokine/Chemokine Panel I Multiplex(Millipore Cat. No. MPXHCYTO-60K; IL-1α, IL-1β, IL-1ra, IL-2, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17,sCD40L, EGF, Eotaxin, FGF-2, G-CSF, GM-CSF, IFN-T, IP-10, MCP-1, MIP-1a,MIP-1β, PDGF-AA, RANTES, TGF-α, TNF-α and VEGF only) and Human GrowthFactor Multiplex (Origene TruPLEX Cat. No. AM100096; PDGF-AB, PDGF-BB,FGF4, VEGFD, FGF2, EGF, HGF, FLT3LG, ANGPT2, PGF and VEGFA).

The ELISA plates were measured using a Molecular Devices UVmax kineticmicroplate reader with SoftMax Pro 5.2 software. The multiplex assayswere performed using the Bio-Rad Bio-Plex system with Bio-Plex Manager4.1 software. Final protein concentrations (pg/mL) were calculated fromthe standard curve for each assay. Depending on the assay, serum samplesmay have been diluted in assay buffer prior to testing. In these cases,protein concentrations were multiplied by the dilution factor.

Results and Discussion:

From 58 patient samples, between 27 and 49 pre- and post-treatment bloodsamples were used for analyses. Significant change in levels of 23factors among 46 factors tested in 50 assays were observed at both, oreither, cycle 1 day8 or cycle 2 day 8 in the serum from patients treatedwith E7080 compared to pre-treatment levels (cycle 1 day 1 (baseline))(Table 9).

TABLE 9 Change in Levels of Blood Biomarkers After E7080 Treatment Timepoint Number Base line Post Treatment Fold change Factor Pre Post PrePost median IQR median IQR median IQR P value EGF CYCLE 1 DAY 1 CYCLE 2DAY 8 42 44 119.9 128.5 75.9 76.6 0.808 0.735 3.70E−02 Eotaxin CYCLE 1DAY 1 CYCLE 1 DAY 8 49 49 128.2 75.9 154.7 117.5 1.171 0.313 3.20E−05CYCLE 1 DAY 1 CYCLE 2 DAY 8 44 44 128.2 75.9 177 123.9 1.363 0.541.10E−06 G-CSF CYCLE 1 DAY 1 CYCLE 1 DAY 8 49 49 51.2 34.5 67.1 46.31.25 0.57 3.80E−04 CYCLE 1 DAY 1 CYCLE 2 DAY 8 44 44 51.2 34.5 61.3 53.81.215 1.011 8.80E−03 IL-6 CYCLE 1 DAY 1 CYCLE 1 DAY 8 37 49 9.9 23.317.3 34.9 1.824 2.759 2.10E−03 CYCLE 1 DAY 1 CYCLE 2 DAY 8 34 44 9.923.3 15 34.4 1.299 2.069 1.90E−02 IL-7 CYCLE 1 DAY 1 CYCLE 1 DAY 8 27 493.3 15.7 6.4 14.5 1 0.507 3.80E−02 IL-8 CYCLE 1 DAY 1 CYCLE 1 DAY 8 4849 26.1 24.9 31.7 28.4 1.164 0.66 1.20E−02 CYCLE 1 DAY 1 CYCLE 2 DAY 843 44 26.1 24.9 31.4 20.3 1.205 0.799 7.80E−03 IL-17 CYCLE 1 DAY 1 CYCLE1 DAY 8 39 49 9.3 30.7 14.4 52.2 1.025 0.378 4.70E−02 IP-10 CYCLE 1 DAY1 CYCLE 1 DAY 8 49 49 387.7 313 474.3 443.1 1.249 0.41 2.70E−05 CYCLE 1DAY 1 CYCLE 2 DAY 8 44 44 387.7 313 602.8 538.5 1.382 0.719 9.40E−04MCP-1 CYCLE 1 DAY 1 CYCLE 2 DAY 8 44 44 582.7 323.3 618 342.1 1.0860.431 4.10E−03 TGFa CYCLE 1 DAY 1 CYCLE 1 DAY 8 45 49 12.2 12 14.6 9.91.157 0.706 2.80E−02 VEGF CYCLE 1 DAY 1 CYCLE 1 DAY 8 48 49 622 1845.8741.4 1918.7 1.29 0.517 1.20E−04 Ang-2 CYCLE 1 DAY 1 CYCLE 1 DAY 8 49 492717 1697.8 1812 1150 0.665 0.298 3.10E−13 CYCLE 1 DAY 1 CYCLE 2 DAY 844 44 2717 1697.8 1533 729 0.618 0.379 1.60E−11 Tie-2 CYCLE 1 DAY 1CYCLE 1 DAY 8 49 49 33675 10817.5 31685 11800 0.917 0.166 9.40E−06 CYCLE1 DAY 1 CYCLE 2 DAY 8 44 44 33675 10817.5 26220 6845 0.766 0.1439.10E−09 sVEGFR1 CYCLE 1 DAY 1 CYCLE 1 DAY 8 43 49 345.2 539.7 300 3410.92 0.361 6.00E−03 CYCLE 1 DAY 1 CYCLE 2 DAY 8 38 44 345.2 539.7 341.5444.9 0.805 0.66 3.50E−02 sVEGFR2 CYCLE 1 DAY 1 CYCLE 1 DAY 8 49 4926778.4 9033.4 18823.1 6482.8 0.676 0.23 7.40E−13 CYCLE 1 DAY 1 CYCLE 2DAY 8 44 44 26778.4 9033.4 13352.1 4041.9 0.499 0.167 5.70E−13 sVEGFR3CYCLE 1 DAY 1 CYCLE 2 DAY 8 39 44 6226.6 4858.8 4030.9 6017.5 0.8430.327 1.80E−03 EGF (80) CYCLE 1 DAY 1 CYCLE 2 DAY 8 38 41 879.6 753.5875.8 823.7 1.17 0.653 4.70E−02 HGF (86) CYCLE 1 DAY 1 CYCLE 1 DAY 8 4648 1201.8 651.4 1339.3 1335.9 1.096 0.572 2.70E−02 FLT3 LG (89) CYCLE 1DAY 1 CYCLE 1 DAY 8 48 48 80.4 56.5 71.1 50.6 0.885 0.249 5.40E−03 ANG2(90) CYCLE 1 DAY 1 CYCLE 1 DAY 8 48 48 928.2 748.9 688.6 442.3 0.6710.218 3.20E−12 CYCLE 1 DAY 1 CYCLE 2 DAY 8 41 41 928.2 748.9 600.6 373.90.586 0.327 2.90E−09 PGF (91) CYCLE 1 DAY 1 CYCLE 1 DAY 8 48 48 16.1 8.535.9 37.2 2.306 1.769 1.70E−09 CYCLE 1 DAY 1 CYCLE 2 DAY 8 41 41 16.18.5 46 55.1 3.05 2.717 3.00E−11 VEGFA (100) CYCLE 1 DAY 1 CYCLE 1 DAY 848 48 125.9 142.4 202.1 204.5 1.28 0.838 9.40E−08 CYCLE 1 DAY 1 CYCLE 2DAY 8 41 41 125.9 142.4 212.5 191.4 1.426 0.941 3.20E−04 SDF-1a CYCLE 1DAY 1 CYCLE 1 DAY 8 49 49 2582.6 957.7 3129 1161.8 1.21 0.172 5.30E−12CYCLE 1 DAY 1 CYCLE 2 DAY 8 43 43 2582.6 957.7 3529.5 970 1.345 0.2466.80E−13

It was next assessed whether changes in expression levels of thesefactors was associated with clinical outcomes (tumor response; PR andothers (SD or PD), tumor shrinkage, and PFS).

TABLE 10 Concentration and Ratio of Blood Biomarkers and Tumor ResponsePR others Factor Time point Median IQR Median IQR p. value IFN-g CYCLE 1DAY 1 7.9 14.1 21.2 60.2 0.013 Ang-2 2530.5 914.5 3399 2288 0.032 ANG2(90) 829.5 541.3 1032.9 1113.4 0.032 ANG2 (90) CYCLE 1 DAY 8 591.7 329.3799.3 505.6 0.043 SDF-1a 3294 1271.3 2915 866.2 0.042 IL-6 CYCLE 2 DAY 88.2 17.8 33.9 33.7 0.026 FGF-2 CYCLE 1 DAY 8/ 1.189 0.821 0.952 0.3970.021 GM-CSF CYCLE 1 DAY 1 1.037 0.641 0.945 0.459 0.044 Eotaxin CYCLE 2DAY 8/ 1.541 0.464 1.242 0.411 0.008 IP-10 CYCLE 1 DAY 1 1.547 0.6161.151 0.606 0.039 Tie-2 0.791 0.159 0.852 0.206 0.023 FGF2 (79) 1.1321.051 0.978 0.391 0.043 GM-CSF CYCLE 2 DAY 8/ 1.051 0.547 0.876 0.4390.07 HGF (86) CYCLE 1 DAY 8 0.789 0.318 1.087 0.491 0.004 VEGFA (100)0.899 0.521 1.172 0.422 0.011Median concentrations of 4 factors (IFN-g, ANG-2, SDF-1a and IL-6) in 5assays at pre-treatment or either 1 week or 5 weeks after treatment withE7080 were significantly different in patients who responded to E7080treatment (PR group) compared with the “others” group (patients with SDor PD) (Table 10, Mann-Whitney U test). For example, concentrations ofIFN-g and ANG-2 at pre-treatment was significantly lower than that seenin patients who had either SD or PD, indicating that low concentrationsof IFN-g and ANG-2 before the commencement of treatment with E7080 arepredictive of a beneficial tumor response to E7080. Changes in theexpression levels of 3 factors (FGF2, Eotaxin, IP-10) were increased inPR group at either cycle1 day8 or cycle2 day8, while the by expressionlevel of these 3 factors were decreased in the “others” groups.Interestingly, GM-CSF expression levels were decreased only in othersgroups at cycle2 day8 compared to either cycle1 day 1 or cycle1 day8. Inaddition, expression levels of Tie-2, HGF, and VEGF were significantlydecreased in PR groups at cycle2 day 8 more than in the “others” groupcompared to cycle 1 day8. These results demonstrated that changes inexpression levels of blood biomarkers were associated with and thereforecan be to predict tumor responses to therapy comprising E7080.

Next, the factors associated with tumor shrinkage were investigated(Pearson's correlation coefficient test, Table 11).

TABLE 11 Concentration and Ratio of Blood Biomarkers and % of MaximumTumor Shrinkage correla- Factor Time point tion (r) p value ANG2(90)CYCLE1 DAY 1 0.309 0.033 PDGF AB(68) −0.420 0.011 Ang-2 0.341 0.012SVEGFR2 0.289 0.036 VEGF 0.276 0.048 IL-10 CYCLE1 DAY 8 −0.315 0.035SDF-1a −0.354 0.009 IL-13 0.399 0.048 PGF(91) CYCLE2 DAY 8 0.305 0.040VEGFA(100) 0.300 0.043 RANTES −0.311 0.045 VEGF 0.292 0.047 FGF-2 CYCLE1DAY 8/ −0.347 0.021 IL-10 CYCLE1 DAY 1 −0.38 0.020 GM-CSF −0.369 0.032IL-1a CYCLE2 DAY 8/ −0.468 0.028 TGFa CYCLE1 DAY 1 −0.348 0.030 IL-6CYCLE2 DAY 8/ 0.392 0.017 Tie-2 CYCLE1 DAY 8 0.352 0.019 PGF(91) −0.3140.046 sVEGFR1 0.332 0.032 VEGFA(100) 0.319 0.035

Concentration of 4 factors (ANG-2, PDGF-AB, sVEGFR2, and VEGF) in 5assays were significantly associated with tumor shrinkage atpre-treatment. These studies indicated that lower concentrations ofthese 4 factors can predict larger tumor shrinkage, while higherconcentration of PDGF-AB might predict larger tumor shrinkage.Concentration of 3 factors (IL-13, PGF, and VEGF) in 4 assays at either1 week or 5 weeks after treatments with E7080 were significantlyassociated with tumor shrinkage. These studies showed that lowerconcentrations of these IL-13, PGF, and VEGF are predictive of largertumor shrinkage at indicated time points. Higher concentration of 3factors (IL-10, SDF1a, and RANTES) in 3 assays at either 1 week or 5weeks after treatments of E7080 are predictive of larger tumorshrinkage. Increase expression levels (indicating high ratio) of FGF2,IL10, GMCSF at cycle1 day8 compared to cycle 1 day1 were significantlyassociated with tumor shrinkage and are predictive of larger tumorshrinkage. At cycle2 day8 compared to cycle1 day 1, a high ratio of IL1aand TGFa is predictive of larger tumor shrinkage. A low ratio of theexpression of 4 factors (IL-6, Tie-2, sVEGFR1, and VEGF) at cycle2 day8compared to cycle1 day8 was associated with larger tumor shrinkage. Ahigh ratio of the expression of PGF at cycle2 day8 compared to cycle 1day8 was associated with larger tumor shrinkage.

Cox proportional hazard model was performed to identify blood biomarkersthat predict progression free survival by either concentrations or ratio(changes in expression levels) of factors. Pharmacokinetic (PK)parameter was used as a covariate in Cox proportional hazards model. Lowconcentrations of ANG-2 and VEGF at cycle 1 day 1, or a low ratio ofIL-12(p40) at cycle2 day8 compared to cycle1 day 1 were significantlyassociated to longer PFS, indicating that these factors can be used asbiomarkers for prediction or response to E7080 therapy (Table 12). Coxproportional hazard model with PK parameter demonstrated that highconcentrations of 7 factors (GCSF, MIP1b, FGF2, MIP1a, IL6, IL13, andsVEGFR3) at pre-treatment can be predictive of longer PFS; whereas, lowconcentrations of ANG-2 at pre-treatment can be predictive of longerPFS. Cox proportional hazard model with PK parameter demonstrated thatlow ratios of 7 factors (FLT3LG, RANTES, GCSF, sVEGFR1, EGF, PDGF-BB,PDGF-AA) are predictive of better PFS and that high ratios of 4 factors(VEGFD, IL10, IL1RA, PDGF-AB) are predictive of better PFS.

TABLE 12 Concentration and Ratio of Blood Biomarker and Progression FreeSurvival p Covariate (p value) Factor Time Coef value MAX1 MAX2 MIN1MIN2 Ang-2 CYCLE1 DAY 1 2.70E−04 0.017 VEGFA (100) 1.40E−03 0.02IL-12(p40) CYCLE 2 DAY8/ 1.90E−01 0.046 CYCLE1 DAY 1 Ang-2 CYCLE1 DAY 13.30E−04 0.004 0.006 0.006 G-CSF −1.60E−02  0.045 0.01 0.007 ANG2 (90)6.50E−04 0.021 0.003 0.01 MIP-1b −1.10E−02  0.048 0.007 0.006 FGF-2−1.10E−02  0.049 0.003 0.004 MIP-1a −2.60E−02  0.033 0.003 0.009 IL-6−3.00E−02  0.018 0.002 0.002 IL-13 −3.00E−02  0.033 0.047 0.049 sVEGFR3−1.00E−04  0.045 0.001 0.003 0.023 FLT3 LG (89) CYCLE1 DAY8/ 1.70E+000.022 0.049 VEGFD (78) CYCLE1 DAY 1 −1.20E+00  0.043 0.006 0.006 RANTES2.50E+00 0.018 0.006 0.014 IL-10 −8.60E−01  0.05 0.014 0.019 IL-1ra−8.30E−01  0.033 0.028 0.008 PDGF AB (68) −2.00E+00  0.043 0.018 0.031G-CSF CYCLE2 DAY8/ 2.20E−01 0.021 0.025 FLT3 LG (89) CYCLE1 DAY 16.40E−01 0.029 0.002 0.008 sVEGFR1 9.30E−01 0.038 0.042 EGF (80)1.10E+00 0.036 0.006 0.01 PDGF BB (73) 4.40E−01 0.044 0.018 PDGF-AA2.30E+00 0.014 0.011 0.003Conclusion:

Concentration and changes in expression levels of cytokines, chemokineand angiogenic factors are associated to tumor response, tumor shrinkageand progression free survival and can be used to predict clinicalresponse to E7080 treatment.

Example 4: Multivariate Analysis to Develop Biomarkers Combining Two orMore than 3 Factors for Prediction of Clinical Outcomes

Purpose:

The purpose of this analysis was to identify combinations of factors,such as mutations, thyroglobulin, blood biomarkers that better associatewith clinical outcomes, such as progression free survival (PFS) than asingle factor and to predict those clinical outcomes.

Materials and Methods:

All factors (i.e., mutations, thyroglobulin, cytokine, chemokine andangiogenic factors) were used as independent variables of interest, thatis, as biomarker candidates. PFS was used as a dependent variable, thatis, as one of the clinical outcomes, in this analysis. Firstly, allfactors were screened according to p-values calculated by Coxproportional hazards model with single factor. Secondly, allcombinations of the screened factors were tested by Cox proportionalhazards model to find significant factors in all combinations of thefactors. Combinations in which all factors were significant were chosenfor further analysis. Hazard functions of the combinations of factorsdefined by their coefficients were obtained from this analysis. Eachpatient has his/her own hazard value for each model, so that patientscan be assigned into two groups when a threshold of hazard is given fora model. After grouping patients, log rank test of progression freesurvival was performed to test difference of survival curves between twogroups (low hazard and high hazard). The best threshold of hazard foreach model of combination of factors was identified by sweepingthreshold to calculate log rank test p-values and finding a thresholdthat minimized the smoothed curve constructed from the p-values, whichwas similar to the approach described in U. Abel, J. Berger and H.Wiebelt, “CRITLEVEL: An Exploratory Procedure for the Evaluation ofQuantitative Prognostic Factors”, Methods Inf. Medicine,23(3):154-6(1984). The best thresholds of the models divided patientsinto high and low hazard groups. Patients are predicted as longer PFSwhen their hazard value is lower than the threshold.

Results and Discussion:

To find biomarkers to predict PFS at pre-treatment, we analyzed thedata, which was available before the treatment. Cox proportional hazardsmodel demonstrated 4 types of combination in two groups of biomarkers.One group is ANG-2, VEGF, GCSF and another group is IL13, MIP1a, andMIP1b. Hazard ratio determined by prediction models indicated thatcombination of VEGF and ANG-2 (Hazard ratio=0.386) predicted PFS betterthan each single factors (VEGF; 0.552, ANG-2; Hazard ratio=0.545).Addition of GCSF to VEGF and ANG-2 did not caused further decrease ofHazard ratio (0.413). Combination of IL13 and MIP1a showed low hazardratio (1.0×10⁻⁹) in our prediction model and addition of MIP1b did notaffect Hazard ratio further (Table 13).

TABLE 13 Predictive Biomarker Using Combination of Blood Biomarkers atPre-Treatment Logrank test N Cox proportional hazards model (p value)Low High p VEGFA ANG-2 GCSF IL13 MIP1a MIP1b Gr Gr value HR* PredictionModel 0.02 15 36 0.198 0.552 (0.0014)*(VEGFA100) − (0.279) < −0.15 0.01727 26 0.108 0.545 (0.000267)*(Ang2) − (0.854) < −0.128 0.037 0.026 23 280.021 0.386 (0.000261)*(Ang2) + (0.00126)*(VEGFA100) − (1.09) < −0.240.016 0.028 0.034 25 26 0.027 0.413 (0.000591)*(ANG290) +(−0.0178)*(GCSF) + (0.00142)*(VEGFA100) − (−0.671) < 0.651 0.009 0.005 718 0.015 1.00E−09 (−0.0459)*(IL13) + (0.0459)*(MIP1a) − (0.0395) < 0.2680.031 0.001 0.049 12 13 0.002 0.121 (−0.0353)*(IL13) +(0.0713)*(MIP1a) + (−0.0154)*(MIP1b) − (0.188) < 0.222

Next we examined if the combination of gene mutation and blood biomarkerpredicted PFS better than gene mutation alone. Cox proportional hazardsmodel demonstrated 3 groups of combination among 3 gene mutations (Table14), such as:

-   -   Group (1): NRAS mutation (mu)    -   a. plus ANG-2;    -   b. plus VEGF, MIP1b;    -   c. plus VEGF, MIP1b, sVEGFR3;    -   d. NRAS mu plus ANG-2, VEGF, MIP1b, sVEGFR3:

Group (2): NRAS mu or KRAS mu plus ANG-2; and

Group (3): NRAS mu or KRAS mu or VHL mu

-   -   a. plus MIP1a;    -   b. plus IL6, VEGF, MIP1a, MIP1b.

For example, hazard ratio determined by the prediction models indicatedthat combination of NRAS mu and ANG-2 (hazard ratio=0.085) predicted PFSbetter than mutation alone (NRAS; hazard ratio=0.124). Also combinationof any of NRAS mu or KRAS mu and ANG-2 (Hazard ratio=0.083) had lowerhazard ratio than mutation only (KRAS mu or NRAS mu; hazardratio=0.205). Combination of any of BRAF mu or NRAS mu or KRAS mu andIL6, VEGF, MIP1a, MIP1b had lower hazard ratio (hazard ratio=1.9E-10)than mutation only (hazard ratio=0.086) (Table 14).

TABLE 14 Predictive Biomarker Using Combination of Gene Mutation andBlood Biomarkers at Pre-Treatment Cox proportional hazards model (pvalue) Logrank test Mutation (any of gene) BRAF, N KRAS, KRAS, Bloodbiomarker Low High p NRAS NRAS NRAS VEGFA ANG-2 MIP1a MIP1b sVEGFR3 IL-6Gr Gr value HR* Prediction Model 0.047 6 17 1.90E−02 0.124 D(NRAS, MU)0.011 0.044 0.035 14 6 1.70E−04 0.097 (−0.025)*log10(MIP1b) +(.0.00616)*log10(VEGFA100) + (3.32)*D(NRAS, WT) − (−0.52) < 1.81 0.0030.01 0.004 0.016 14 6 8.70E−06 0.054 (−0.0494)*log10(MIP1b) +(−0.000472)*log10(sVEGFR3) + (−0.0119)*log10(sVEGFA100) + (4.66)*D(NRAS,WT) − (.5.9) < 3.55 0.003 0.006 0.017 0.007 0.005 12 8 2.00E−06 2.60E−10(0.00148)*log10(Ang2) + (−0.0606)*log10(MIP1b) +(−0.000917)*log10(VEGFR3) + (−0.0177)*log10(VEGFA100) + (6.58)*D(NRAS,WT) − (−5.78) < 3.97 0.015 0.049 13 8 5.10E−04 0.115(0.000751)*log10(ANg2) + (2.69)*D(NRAS, WT) − (3.92) < 0.716 0.013 0.02814 7 2.80E−04 0.085 (0.000972)*log10(Ang290) + (2.75)*D(NRAS, WT) −(2.96) < 0.633 0.043 8 15 2.70E−02 0.205 D(KRASNRAS, MU) 0.01 0.044 13 82.20E−04 0.083 (0.000869)*log10(ANG290) + (2.16)*D(KRASNRAS, WT) −(2.24) < 0.508 0.004 0.044 11 9 2.60E−04 0.086 (−0.0281)*log10(MIP1a) +(2.19)* D(BRAFKRASNRAS, WT) − (−0.41) < −0.0348 0.01 0.017 0.015 0.0160.019 10 5 3.30E−05 1.90E−10 (0.126)*log10(IL6) +(−0.193)*log10(MIP1a) + (−0.0775)*log10(MIP1b) +(−0.0514)*log10(VEGFA100) + (7.94)*D(BRAFKRASNRAS, WT) − (−14.4) < 4.69Conclusion:

Combination of biomarkers, either gene mutations or blood biomarkers orcombination among blood biomarkers predicted PFS better than as a singlebiomarker based on prediction models after determining combinations ofthem using the Cox proportional hazard model. Biomarker combinationsthat were found by this analysis may be used to predict clinicaloutcomes such as PFS with E7080 and response to E7080 treatment.

Example 5: Mutation Status as Predictive Biomarkers for RCC Patients'Responsiveness to Therapy Comprising E7080 Either Alone or inCombination with Everolimus

Purpose:

Tumor response and prolonged disease stabilization are observed in renalcell carcinoma patients (RCC) treated in a phase II study with E7080(methanesulfonic acid salt of lenvatinib) alone or in combination withEverolimus. This experiment is directed at identifying amino acidmutations that are useful in predicting whether RCC subjects respond, orfail to respond, to treatment with E7080 using three criteria ofresponse: best overall response, tumor shrinkage, and progression freesurvival.

Materials and Methods:

Tissue samples are obtained at surgery before the patients had receivedany therapy comprising E7080 and were routinely processed with formalinfixed, paraffin embedded tissues (FFPE). The protocol that is used isapproved by the institutional review board, and informed consent isobtained from each subject. Tumor tissue samples from X patients, forwhich tissues are available, will be used for mutation analysis. DNA isisolated from FFPE tumor blocks collected from patients participating inthe trial. Genomic DNA is extracted from two to five 10 micron unstainedsections by deparaffinization and Qiagen DNA Mini Kit Tissue Protocolwith minor modification. For mutation detection, a sequencing technologysuch as the SEQUENOM® (San Diego, Calif.) platform and the OncoCarta™Panel v1.0 and OncoCarta™ Panel v3.0 described in Example 1,semiconductor sequencing such as Ion Torrent PGM, High Resolution MeltAnalysis, or classical Sanger sequencing is used.

The period during which a patient takes E7080 is artificially dividedinto different Cycles for ease of evaluation and tracking. Patientsreceive E7080 at a dose of 24 mg orally once daily in 28 day cycleseither alone or in combination with everolimus (E7080 and/oreverolimus). For the E7080 Renal cell Carcinoma trial, each Cycle is 28days (4 weeks) so Day 1-28 is cycle 1: Day 29 is Day 1 of Cycle 2; andDay 57 is Day 1 of Cycle 3. Blood samples are collected forpharmacokinetic (PK) analysis on Cycle 1 Days 1, Cycle 2 Day 1, andCycle 3 Day 1. A total of 6 samples per patient are collected asfollows: Cycle 1 Day 1: immediately prior to the dose of E7080, and 2 to8 hours following the first dose of E7080 (post-dose); Cycle 2 Day 1:immediately prior to the dose of E7080 and 2 to 8 hours following thefirst dose of E7080 (post-dose); Cycle 3 Day 1: immediately prior to thedose of E7080 and 2 to 8 hours following the first dose of E7080(post-dose). For analysis of progression free survival, PK parameter isused as a covariate in Cox proportional hazards model.

The four criteria of response: best overall response, tumor response,tumor shrinkage, and progression free survival are defined below.

“Best Overall Response” (BOR) refers to having one of the followingstatuses—Complete Response (CR), Partial Response (PR), Stable Disease(SD) or Progressive Disease (PD) an association of BOR to gene mutationis analyzed by Fisher's exact test.

“Clinical benefit” (CB) refers to having one of the followingstatuses—Complete Response (CR), Partial Response (PR); or StableDisease (SD) with 6 months or more progression free survival (PFS).

“Complete Response” means complete disappearance of all target lesions.

“Partial Response” means at least 30% decrease in the sum of the longestdiameter (LD) of target lesions, taking as reference the baseline summedLD.

“Progressive Disease” (PD) means at least 20% increase in the sum of theLD of target lesions, taking as reference the smallest summed LDrecorded since the treatment started, or the appearance of one or morenew lesions.

“Stable Disease” means neither sufficient shrinkage of the targetlesions to qualify for PR nor sufficient increase to qualify forprogressive disease (PD), taking as reference the smallest summed LDsince the treatment started.

“Progression Free Survival” (PFS) refers to the period from start dateof treatment to the last date before entering PD status and correlationof gene mutation to PFS is analyzed by Logrank test and Cox proportionalhazards model.

“Tumor shrinkage” (TS) means percent change of sum of diameters oftarget lesions, taking as reference the baseline sum diameters andcorrelation of gene mutation to TS is analyzed by Pearson product-momentcorrelation coefficient and Spearman's rank correlation coefficienttest.

“Tumor response” (TR) compares subjects with “Partial Response” (PR)with subjects with either Stable Disease (SD) or Progressive Disease(PD). ⇒not for mutation analysis, but for TG and blood biomarkers.

Example 6: Blood Biomarkers as Predictive and Response Biomarkers ofE7080 Either Alone or in Combination with Everolimus in RCC Subjects

Purpose:

The purpose of this analysis is to measure cytokine, chemokine andangiogenic factors (collectively referred to herein as “bloodbiomarkers”) in blood samples obtained from patients in clinical trialsat both pre- and post-treatment with E7080 and/or everolimus and toidentify blood biomarkers which can be used to predict whether renalcell carcinoma patients will respond or fail to respond to treatmentwith E7080. For these analyses, four criteria of response are employed,namely: best overall response, tumor response, % of tumor shrinkage andprogression free survival.

Materials and Methods:

Patients receive E7080 at a dose of 24 mg oral once daily in 28 daycycles either alone or in combination with everolimus. Serum samples arecollected at Cycle 1 Day 1 (pre-treatment), Cycle 1 Day 15 andimmediately before dosing on Day1 of each subsequent cycle and at thetime when the patient is off-treatment. 7.5 mL of blood is drawn intoserum stainless steel tube (SST) and let to clot at room temperature forat least 30 min. Within 60 minutes of sample collection, the tubes arecentrifuged for 10 minutes at 20-25° C. at 1400×g. The supernatant isdrawn without disturbing the pellet and the serum is mixed and dividedinto two sample tubes and stored frozen (−70° C.) at an upright positionuntil further treatment for analysis. Serum samples from patients areused for blood biomarker analysis. Serum from Cycle 1 day 1 (baseline),cycle 1 day 15, pre-dose sample from Day1 of each subsequent cycle andserum sample when the patient comes off treatment are used in thisanalysis. The association of progression free survival with or without acovariate is analyzed. The covariates used are the following PKparameters: cycle 1 day 1 Cmax (E7080 concentration 2 hrs to 8 hrs afterdosing on cycle 1 day 1: MAX1); cycle2 day 1 Cmax (E7080 concentration 2hrs to 8 hrs after dosing on cycle2 day 1:MAX2); cycle2 day 1 Ctrough(E7080 concentration before dosing on cycle 2 day 1:MIN2). Serum samplesare tested in batch format where all timepoints from the same subjectare assayed on the same day. On the day of assay, samples are removedfrom −80° C. and allowed to thaw and reach room temperature. The serumsamples are tested using the following commercial assay kits as per themanufacturer's instructions: Human Soluble Tie-2 ELISA (R&D Systems Cat.No. DTE200), Human Angiopoietin-1 ELISA (R&D Systems Cat. No. DANG10),Human Angiopoietin-2 ELISA (R&D Systems Cat. No. DANG20), Human SolubleReceptor Multiplex (Millipore Cat. No. HSCR-32K; sVEGF R1, sVEGF R2 andsVEGF R3 only), Human Cytokine/Chemokine Panel I Multiplex (MilliporeCat. No. MPXHCYTO-60K; IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6,IL-7, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, sCD40L, EGF,Eotaxin, FGF-2, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β,PDGF-AA, RANTES, TGF-α, TNF-α and VEGF only) and Human Growth FactorMultiplex (Origene TruPLEX Cat. No. AM100096; PDGF-AB, PDGF-BB, FGF4,VEGFD, FGF2, EGF, HGF, FLT3LG, ANGPT2, PGF and VEGFA).

The ELISA plates are measured using a Molecular Devices UVmax kineticmicroplate reader with SoftMax Pro 5.2 software. The multiplex assaysare performed using the Bio-Rad Bio-Plex system with Bio-Plex Manager4.1 software. Final protein concentrations (pg/mL) are calculated fromthe standard curve for each assay. Depending on the assay, serum samplesmay be diluted in assay buffer prior to testing. In these cases, proteinconcentrations are multiplied by the dilution factor.

The four criteria of response; best overall response, tumor response,tumor shrinkage, and progression free survival are defined below andanalyzed by indicated methods.

“Best Overall Response” (BOR) refers to having one of the followingstatuses: —Complete Response (CR), Partial Response (PR), Stable Disease(SD) or Progressive Disease (PD) and association of BOR to gene mutationis analyzed by Fisher's exact test

“Clinical benefit” (CB) refers to having one of the following statusesand association of CB to gene mutation is analyzed by student's t-testand Mann-Whitney U test.

-   -   Complete Response (CR), Partial Response (PR); or Stable Disease        (SD) with 6 months or more progression free survival (PFS)

“Complete Response” means complete disappearance of all target lesions.

“Partial Response” means at least 30% decrease in the sum of the longestdiameter (LD) of target lesions, taking as reference the baseline summedLD.

“Progressive Disease” (PD) means at least 20% increase in the sum of theLD of target lesions, taking as reference the smallest summed LDrecorded since the treatment started, or the appearance of one or morenew lesions.

“Stable Disease” means neither sufficient shrinkage of the targetlesions to qualify for PR nor sufficient increase to qualify forprogressive disease (PD), taking as reference the smallest summed LDsince the treatment started.

“Progression Free Survival” (PFS) refers to the period from start dateof treatment to the last date before entering PD status and correlationof gene mutation to PFS is analyzed by Logrank test and cox proportionalhazards model.

“Tumor shrinkage” (TS) means percent change of sum of diameters oftarget lesions, taking as reference the baseline sum diameters andcorrelation of gene mutation to TS is analyzed by Pearson product-momentcorrelation coefficient and * Spearman's rank correlation coefficienttest.

“Tumor response” (TR) compares subjects with “Partial Response” (PR)with subjects with either Stable Disease (SD) or Progressive Disease(PD) and association of blood biomarkers is analyzed by student's t-testand Mann-Whitney U test.

Example 7: Multivariate Analysis to Develop Biomarkers Combining Two orMore than 3 Factors for Prediction of Clinical Outcomes in RCC Patients

Purpose:

The purpose of this analysis is to identify combinations of factors,such as mutations, and levels or expression ratios of cytokine,chemokine and angiogenic factors, that better associate with progressionfree survival (PFS) than a single factor and to predict clinicaloutcomes, such as PFS and TS, in RCC patients.

Materials and Methods:

All factors (i.e., mutations, cytokine, chemokine and angiogenicfactors) are used as independent variables of interest, that is, asbiomarker candidates. PFS is used as a dependent variable, that is, as aclinical outcome in this analysis. Firstly, all factors are screenedaccording to p values calculated by Cox proportional hazards model withsingle factor. Secondly, all combinations of the screened factors aretested by Cox proportional hazards model to find significant factors inall combinations of the factors. Combinations in which all factors aresignificant are chosen for further analysis. Hazard functions of thecombinations of factors defined by their coefficients are obtained fromthis analysis. Each patient has his/her own hazard value for each model,so that patients can be assigned into two groups when a threshold ofhazard is given for a model. After grouping patients, log rank test ofprogression free survival is performed to test difference of survivalcurves between two groups (low hazard and high hazard). The bestthreshold of hazard for each model of combination of factors isidentified by sweeping threshold to calculate log rank test p-values andfinding a threshold that minimizes the smoothed curve constructed fromthe p-values, which is similar to the approach described in U. Abel, J.Berger and H. Wiebelt, “CRITLEVEL: An Exploratory Procedure for theEvaluation of Quantitative Prognostic Factors”, Methods Inf. Medicine,23(3):154-6(1984). The best thresholds of the models divides patientsinto high and low hazard groups. The criteria of patient prediction oflonger PFS are defined as a hazard value calculated by hazard functionthat is lower than the threshold.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method for treating differentiated thyroidcancer, comprising: providing a first blood sample obtained, beforeinitiation of a therapy comprising lenvatinib or a pharmaceuticallyacceptable salt thereof, from a human subject having or suspected ofhaving a differentiated thyroid cancer; providing a second blood sampleobtained from the human subject after initiation of the therapycomprising lenvatinib or a pharmaceutically acceptable salt thereof;measuring the concentration of thyroglobulin in the first blood sampleand the second blood sample, wherein the ratio of the concentrations ofthyroglobulin in the blood samples (second blood sample/first bloodsample) is reduced as compared to a control, wherein the control is apre-established reference ratio established by an analysis ofthyroglobulin expression pre- and post-treatment with lenvatinib or apharmaceutically acceptable salt thereof in one or more subjects thathave not responded to treatment with lenvatinib or a pharmaceuticallyacceptable salt thereof; and further administering lenvatinib or apharmaceutically acceptable salt thereof to the human subject, whereinthe human subject has a ratio of the concentrations of thyroglobulin inthe blood samples (second blood sample/first blood sample) that isreduced as compared to the control.
 2. The method of claim 1, whereinthe differentiated thyroid cancer is a follicular thyroid cancer.
 3. Themethod of claim 1, wherein the differentiated thyroid cancer is apapillary thyroid cancer.
 4. The method of claim 1, wherein thelenvatinib or pharmaceutically acceptable salt thereof is lenvatinibmesylate.